Petra Krause

Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells∗

BACKGROUND/AIMS A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h. This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days. METHODS Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen. RESULTS At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v). Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h. The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein. CONCLUSIONS The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (α-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Liver repopulation after hepatocellular transplantation: integration and interaction of transplanted hepatocytes in the host.

The mechanisms of donor hepatocyte integration into recipient liver are not fully understood. We investigated mechanisms of both the integration and interaction of transplanted hepatocytes with host liver cells as well as the repopulation of the host organ following intraportal transplantation. Mature hepatocytes were injected into the portal vein of dipeptidylpeptidase IV (DPPIV)-deficient rats pretreated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. The degree of integration and proliferation was studied by colocalizing transplanted cells (DPPIV positive) with connexin 32, MMP-2, and OX-43 (multilayer immunofluorescence imaging). FACS analysis was established to assess the extent of repopulation quantitatively. Transplanted hepatocytes reached the distal portal spaces and sinusoids within 1 h after injection. A small proportion of cells succeeded in traversing the endothelial barrier through mechanical disruption in both locations. Transplanted hepatocytes lost their membrane-bound gap junctions (connexin 32) during this process. Successful integration of the donor cells required up to 5 days, heralded by gap junction reconstitution and the specific basolateral membrane expression of DPPIV. MMP-2 degraded the extracellular matrix in close proximity to donor cells, providing space for cell division. FACS analysis revealed that more than 37% of the liver was repopulated by cells derived from donors at 2 months after transplantation. Our data demonstrate a high degree of donor cell repopulation of the host organ and provide valuable insight into the specific mechanisms of donor cell integration. Connexin 32 expression in transplanted hepatocytes may serve as an indicator of their effective incorporation and communication within the recipient liver. FACS analysis reveals an accurate method to determine quantitatively the extent of liver repopulation.

Irradiation as preparative regimen for hepatocyte transplantation causes prolonged cell cycle block.

Purpose: Hepatocyte transplantation following liver irradiation (IR) and partial hepatectomy (PH) leads to extensive liver repopulation. We investigated the changes in the liver induced by IR explaining the loss of reproductive integrity in endogenous hepatocytes. Materials and methods: Right lobules of rat liver underwent external beam IR (25 Gy). A second group was subjected to additional 33% PH of the untreated left liver lobule. Liver specimens and controls were analyzed for DNA damage, apoptosis, proliferation and cell cycle related genes (1 hour to up to 12 weeks). Results: Double strand breaks (phosphorylated histone H2AX) induced by IR rapidly declined within hours and were no longer detectable after 4 days. No significant apoptosis was noted and steady mRNA levels (B-cell lymphoma 2-associated X protein (BAX), caspase 3 and 9) were in line with the lack of DNA fragmentation. However, gene expression of p53 and p21 in irradiated liver tissue increased. Transcripts of cyclin D1, proliferating cell nuclear antigen (PCNA), and cyclin B augmented progressively, whereas cyclin E was only affected moderately. Following PH, irradiated livers displayed persistently high protein levels of p21 and cyclin D1. However, cell divisions were infrequent, as reflected by low PCNA levels up to four weeks. Conclusion: IR leads to a major arrest in the G1/S phase and to a lesser extent in the G2/M transition of the cell cycle, resulting in reduced regenerative response following PH. The persistent block of at least four weeks may promote preferential proliferation of transplanted hepatocytes in this milieu.

Regional transient portal ischemia and irradiation as preparative regimen for hepatocyte transplantation.

Hepatocyte transplantation is regarded as a promising option to correct hereditary metabolic liver disease. This study describes a novel method involving regional transient portal ischemia (RTPI) in combination with hepatic irradiation (IR) as a preparative regimen for hepatocyte transplantation. The right lobules of rat livers (45% of liver mass) were subjected to RTPI of 30–120 min. Liver specimens and serum samples were analyzed for transaminase levels, DNA damage, apoptosis, and proliferation. Repopulation experiments involved livers of dipeptidylpeptidase IV (DPPIV)-deficient rats preconditioned with RTPI (60–90 min) either with or without prior partial hepatic IR (25 Gy). After reperfusion intervals of 1 and 24 h, 12 million wild-type (DPPIV positive) hepatocytes were transplanted into recipient livers via the spleen. RTPI of 60–90 min caused limited hepatic injury through necrosis and induced a distinct regenerative response in the host liver. Twelve weeks following transplantation, small clusters of donor hepatocytes were detected within the portal areas. Quantitative analysis revealed limited engraftment of 0.79% to 2.95%, whereas control animals (sham OP) exhibited 4.16% (determined as relative activity of DPPIV when compared to wild-type liver). Repopulation was significantly enhanced (21.43%) when IR was performed prior to RTPI, optimum preconditioning settings being 90 min of ischemia and 1 h of reperfusion before transplantation. We demonstrate that RTPI alone is disadvantageous to donor cell engraftment, whereas the combination of IR with RTPI comprises an effective preparative regimen for liver repopulation. The method described clearly has potential for clinical application.

Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model.

SummaryThe specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4–14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.

Functional characterization of serum-free cultured rat hepatocytes for downstream transplantation applications.

Although ex vivo culture of hepatocytes is known to impair functionality, it may still be considered as desirable to propagate or manipulate them in culture prior to transplantation into the host liver. The aim of this study was to clarify whether rat hepatocytes cultured over different periods of time proliferate and retain their hepatocyte-specific functions following transplantation into the recipient liver. Rat hepatocytes were cultured under serum-free conditions in the presence of hepatocyte and epidermal growth factors. Cells derived from wild-type donor livers were transplanted into the livers of CD26-deficient rats. Cell proliferation and the expression of hepatocyte-specific markers were determined before and after transplantation. Cell number increased threefold over a culture period of 10 days. The expression of connexin 32 and phosphoenolpyruvate carboxykinase declined over time, indicating the loss of hepatocyte-specific functions. Hepatocytes cultured over 4 or 7 days and then transplanted proliferated in the host parenchyma. The transplanted cells expressed connexin 32, cytokeratin 18, and phosphoenolpyruvate carboxykinase, indicating the differentiated phenotype. The loss of hepatocyte-specific functions during culture may be restored after transplantation, suggesting that the proper physiological environment is required to maintain the differentiated phenotype.

Fractionated External Beam Radiotherapy as a Suitable Preparative Regimen for Hepatocyte Transplantation After Partial Hepatectomy

PURPOSE Hepatocyte transplantation is strongly considered to be a promising option to correct chronic liver failure through repopulation of the diseased organ. We already reported on extensive liver repopulation by hepatocytes transplanted into rats preconditioned with 25-Gy single dose selective external beam irradiation (IR). Herein, we tested lower radiation doses and fractionated protocols, which would be applicable in clinical use. METHODS AND MATERIAL Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with partial liver external beam single dose IR at 25 Gy, 8 Gy, or 5 Gy, or fractionated IR at 5 × 5 Gy or 5 × 2 Gy. Four days after completion of IR, a partial hepatectomy (PH) was performed to resect the untreated liver section. Subsequently, 12 million wild-type (DPPIV(+)) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor cell integration and liver repopulation was studied 16 weeks after transplantation by means of immunofluorescence and DPPIV-luminescence assay. RESULTS Donor hepatocyte integration and liver repopulation were more effective in the irradiated livers following pretreatment with the IR doses 1 × 25 Gy and 5 × 5 Gy (formation of large DPPIV-positive cell clusters) than single-dose irradiation at 8 Gy or 5 Gy (DPPIV-positive clusters noticeably smaller and less frequent). Quantitative analysis of extracted DPPIV revealed signals exceeding the control level in all transplanted animals treated with IR and PH. Compared with the standard treatment of 1 × 25 Gy, fractionation with 5 × 5 Gy was equally efficacious, the Mann-Whitney U test disclosing no statistically significant difference (p = 0.146). The lower doses of 1 × 5 Gy, 1 × 8 Gy, and 5 × 2 Gy were significantly less effective with p < 0.05. CONCLUSION This study suggests that fractionated radiotherapy in combination with PH is a conceivable pretreatment approach to prime the host liver for hepatocyte transplantation, thus bringing the experimental model a step closer to clinical application.

Role of carbon monoxide and nitric oxide in adult rat hepatocytes proliferating in vitro: Effects of CAS 1609.

During liver regeneration in vivo carbon monoxide (CO) and nitric oxide (NO) are supposed to play a significant role. We raise the question whether CO and NO are involved in the growth process of cultured hepatocytes. Rat hepatocytes were stimulated into proliferation, growth being estimated by DNA content, mRNA by quantitative RT-PCR, and inducible NO synthase (iNOS) activity by GC-MS. Dexamethasone proved obligatory for fast proliferation. It suppressed the spontaneous rise of iNOS-mRNA in cultures devoid of glucocorticoids, but did not counteract the rise in mRNA in actively dividing cultures. Expression of iNOS-mRNA and cell growth were further enhanced by LiCl (10 mM). NOS activity was completely suppressed by the iNOS-specific inhibitors N-(3-(aminomethyl)benzyl) acetamidine (1400 W,100 microM) and L-N(6)-(1-iminoethyl)lysine (L-NIL, 500 microM), however, without a decrease in hepatocyte growth. Proliferation was attenuated only by very high concentrations (>0.5 mM) of N-nitro-L-arginine methyl ester (L-NAME) and asymmetric dimethylarginine (ADMA). Various NO donors (at 100 microM) did not stimulate cell growth. The furoxan CAS 1609 stimulated growth, decreased iNOS-mRNA expression and transiently increased haem oxygenase-1 (HO-1)-mRNA without releasing considerable amounts of NO. 1H-[1,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ) attenuated the action of CAS 1609. Proliferation was stimulated by Co-protoporphyrin and tricarbonyldichlororuthenium(II) dimer (CORM-2). We conclude that CAS 1609 triggers hepatocyte mitosis most likely via direct, NO-independent induction of HO-1 expression, pointing to CO as a growth-promoting signal in the proliferation cascade in cultured hepatocytes.

Allogeneic hepatocyte transplantation using fk 506

Hepatocyte transplantation is an intriguing alternative to orthotopic liver transplantation. While engraftment of syngeneic hepatocytes can be achieved with relative ease, engraftment of allogeneic hepatocytes has been far more complicated. We used FK 506 (Tacrolimus), a novel and highly efficient immunosuppressant, which has been reported to augment liver regeneration in rats. Recipients of isolated syngeneic (LEW) and allogeneic (Wistar F.) rat hepatocytes (major histocompatibility barrier) recieved different immunosuppressive regiments with FK 506 or Cyclosporine A (CsA). Mature syngeneic hepatocytes could be retrieved up to post op day 300 with the lowest number of hepatocytes on post op day 20. Following allogeneic transplantation, no mature hepatocytes could be identified after post op day 10, though ductular like structures within the spleen were found in FK 506 but not CsA-treated animals. The epithelial cells of ductular like structures exhibit cytological features of CK-19 positive cells. Our results suggest that under CsA or FK 506 immunosuppression long-term survival of mature allogeneic hepatocytes within the spleen cannot be achieved across a major histocompatibility barrier though FK 506 allows engraftment of allogeneic donor type ductular cells.