Enhanced proliferation of hepatic progenitor cells in rats after portal branch occlusion
2004; Lippincott Williams & Wilkins; Volume: 10; Issue: 6 Linguagem: Inglês
10.1002/lt.20156
ISSN1527-6473
AutoresNorihito Ise, Tsutomu Sato, Ouki Yasui, Go Watanabe, Kenji Koyama, Kunihiko Terada, Toshihiro Sugiyama, Yuzo Yamamoto,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoLiver TransplantationVolume 10, Issue 6 p. 748-754 Original ArticleFree Access Enhanced proliferation of hepatic progenitor cells in rats after portal branch occlusion Norihito Ise, Norihito Ise Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorTsutomu Sato, Corresponding Author Tsutomu Sato [email protected] Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, Japan Telephone: +81-18-884-6215; FAX: +81-18-836-2614Department of Gastroenterological Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, JapanSearch for more papers by this authorOuki Yasui, Ouki Yasui Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorGo Watanabe, Go Watanabe Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorKenji Koyama, Kenji Koyama Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorKunihiko Terada, Kunihiko Terada Department of Biochemistry, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorToshihiro Sugiyama, Toshihiro Sugiyama Department of Biochemistry, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorYuzo Yamamoto, Yuzo Yamamoto Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this author Norihito Ise, Norihito Ise Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorTsutomu Sato, Corresponding Author Tsutomu Sato [email protected] Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, Japan Telephone: +81-18-884-6215; FAX: +81-18-836-2614Department of Gastroenterological Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, JapanSearch for more papers by this authorOuki Yasui, Ouki Yasui Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorGo Watanabe, Go Watanabe Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorKenji Koyama, Kenji Koyama Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorKunihiko Terada, Kunihiko Terada Department of Biochemistry, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorToshihiro Sugiyama, Toshihiro Sugiyama Department of Biochemistry, Akita University School of Medicine, Akita, JapanSearch for more papers by this authorYuzo Yamamoto, Yuzo Yamamoto Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, JapanSearch for more papers by this author First published: 20 May 2004 https://doi.org/10.1002/lt.20156Citations: 3 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract It is known that hepatic progenitor cells increase in number after liver injury caused by carcinogens, but this injury cannot be reproduced in humans. In order to create a practical source of hepatic progenitor cells, changes in the number of liver epithelial cells (LECs), a type of hepatic progenitor cell, were examined following partial interruption of the portal flow. Efficiency in this isolation procedure was investigated, and isolated LECs were transplanted into livers to demonstrate their differentiation into hepatocytes in vivo.A volume of 70% of Sprague-Dawley rat's livers was exposed to portal vein ligation. LECs, identified as alpha-fetoprotein (AFP)-positive and albumin-negative cells, were counted and LECs isolated from the portal vein ligated-lobe were characterized by immunostaining and Western blotting. Isolated cells were subjected to a 1-week-culture, and the number of colonies formed on dishes was counted. The cells were then transplanted to the livers of genetic analbuminemic rats and identified by immunohistochemistry. The number of LECs in the portal ligated-lobes on day 7 was 14.7 ± 6.5 cells/1,000 hepatocytes: 18 times higher than numbers in a normal liver. A significant increase was noted from day 3 until day 28. Isolated LECs were AFP-positive, albumin-negative, and cytokeratin-19–positive cells. The number of colonies on the 7th day following portal vein ligation was 42 times higher than in a normal liver. After transplantation of the LECs to the analbuminemic rat, a cluster of albumin-producing cells was present until day 56, suggesting that they differentiate into hepatocytes. We conclude that after portal vein occlusion, the liver can be a good source of hepatic progenitor cell. These results open up the possibility of cellular transplantation for liver functional support in clinical settings. (Liver Transpl 2004;10:748–754.) A shortage of hepatic functional capacity after extended liver resection often results in a systemic metabolic imbalance of organs or even hepatic failure.1, 2 Although various strategies have been developed to minimize the decrease in functional reserve after liver surgery,3-5 none of them have offered a practical solution. Hepatocyte transplantation is also one of the treatment modalities,6-8 but remains impractical, chiefly because sufficient amounts of viable liver cells are not always available for this purpose. On the other hand, cellular transplantation using stem cells or progenitor cells has attracted much attention because progenitor cells can be multiplied in vitro and can solve the problem of shortage in cell numbers. Moreover, it has been acknowledged that certain kinds of liver progenitor cells can differentiate into hepatocytes in vivo, and thus enhance the function as well as regenerative ability of the liver.9-11 With regard to liver progenitor cells, there is still considerable confusion in the definition and nomenclature of the cells.12 Liver progenitor cells can be defined as ones that show a bidirectional differentiation into hepatocytes and bile duct cells, and have a self-replication ability. Oval cells, liver epithelial cells (LECs), and small hepatocytes are thought to be heterogeneous subpopulations of liver progenitor cells, but there could be overlaps among them with respect to their phenotypic features. We have previously reported that oval cells isolated from Long-Evans Cinnamon rats could differentiate into hepatocytes in vivo13 and that differentiation of liver epithelial (stem-like) cells into hepatocytes in vitro could be induced in a coculture with hepatic stellate cells.14 Such progenitor cells obtained from host livers would be an ideal choice for hepatocyte transplantation into remnant livers after liver surgery to augment hepatic functional capacity, because immunological reactions would be eliminated. However, one of the obstacles in using progenitor cells for this purpose is that, while they certainly exist, their number in the normal liver is very small. Recently, a marked increase of progenitor cells such as oval cells has been reported in injured livers following exposure to chemical carcinogens or a special diet (e.g., a choline-deficient diet).15, 16 Although this is very attractive as a possible rich source of progenitor cells, the use of carcinogens or a special diet is not appropriate in the clinical setting. Given that the increase of progenitor cells in the presence of chemical carcinogens is closely related to hepatocyte damage or to a decrease in the number of viable hepatocytes, it is very likely that local hepatic ischemia might also contribute to an increase in progenitor cells. Therefore, it is intriguing to investigate whether the number of progenitor cells increases in hepatic lobes after partial liver ischemia or portal vein occlusion. In this study, we investigated whether the LECs, established hepatic progenitor cells, proliferated in the hepatic lobes after hemiligation of portal veins, and whether isolating LECs from livers after portal vein ligation was efficient. In addition, we also examined isolated LECs transplanted to the livers of Nagase analbuminemic rats (NARs) to evaluate the function of the cells. Abbreviations LECs, liver epithelial cells; AFP, alpha-fetoprotein; NARs, Nagase analbuminemic rats. Materials and Methods Animal Preparation and Liver Procurement All experiments were conducted in compliance with the Institutional Guidelines regarding Animal Research at Akita University School of Medicine, which were based on the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Eight-week-old female Sprague-Dawley rats (Charles River Japan, Osaka, Japan) were used. After inhalation anesthesia using diethyl ether, abdomens were opened and left portal vein branches were exposed and ligated. Animals were sacrificed on days 1, 3, 5, 7, 9, 11, 14, 21, and 28 following portal vein ligation (n = 4 at each time point). Livers were procured, and ligated lobes and nonligated lobes were fixed in 10% formaldehyde solution and embedded in paraffin. Serial liver tissue sections underwent light microscopic observation after hematoxylin-eosin, and/or immunohistochemical staining. Immunohistochemistry After blocking the activity of endogenous peroxidase with 0.3% H2O2, liver sections were prepared for antigen retrieval and incubated with antibodies against AFP (goat polyclonal IgG anti-human AFP, diluted at 1:150; Santa Cruz, CA), albumin (rabbit anti-rat albumin, diluted at 1:500; Inter-Cell Technologies, Hopewell, NJ) at room temperature for 60 minutes. Sections were then incubated with secondary biotinylated antibodies diluted to 1:250 in phosphate buffer solution containing 1% bovine serum albumin at room temperature for 30 minutes. The reaction was followed by incubation with avidin-biotinylated horseradish peroxidase (Vector Laboratories, Burlingame, CA). The sections were then developed in a solution containing 0.1% diaminobenzidine tetrahydrochloride, 0.01% H2O2, and 0.1M Tris-HCl. Finally, the sections were counterstained with hematoxylin. Both in the ligated and nonligated lobes, 1,000 liver cells with nuclei were counted from 5 randomly selected fields under a light microscope. AFP-positive and albumin-negative cells were counted as LECs and the number of LECs per the total number of liver cells was expressed in parts per 1,000. To examine proliferative activity of LECs appearing in the liver, serial sections following AFP staining were prepared and subjected to immunostaining of Ki-67 antigen (Dako Cytomation, Glostrup, Denmark) using the same procedure as described above. Isolation and Purification of LECs Another dozen Sprague-Dawley rats were used. The left branches of the portal veins were ligated as described above. Rats were sacrificed on days 3, 7, or 14 following portal vein ligation (n = 4 at each time point). Rats sacrificed without portal vein ligation served as controls (n = 4). LECs were isolated from the ligated lobes as described elsewhere.13 Briefly, isolated liver cells were prepared using a 2-step collagenase perfusion via the inferior vena cava, and liver cell suspensions were treated at 37°C for 60 minutes with 0.1% protease E (Kaken Pharmaceuticals, Tokyo, Japan) and 0.25% trypsin. Cells were then centrifuged at 2,000 rpm (15-cm radius) for 20 minutes on a discontinuous gradient of Percoll (Pharmacia, Uppsala, Sweden) at a density of 1.035 and 1.070 gm/mL. The viability of isolated cells was confirmed by Trypan blue dye exclusion test as 92%. After adjusting the density of cell suspensions in Roswell Park Memorial Institute medium (RPMI-1640; Nissui Pharmaceutical, Tokyo, Japan) containing immobilized 10% fetal calf serum (Sigma Chemical, St. Louis, MO), cells were seeded onto 60-mm type IV collagen-coated dishes (Falcon, Franklin Lakes, NJ) at a density of 7.5 × 104/dish and cultured for 1 week at 37°C in an atmosphere of 95% O2 and 5% CO2. After 1 week in this culture, LECs were identified as the colony-forming subpopulation of the isolated cells, and other types of cells were removed from this culture as described elsewhere.17 The proliferated cells were then immersed in a cell-preserving liquid with dimethyl sulfoxide (DMSO; Zenoaq, Kouriyama, Japan) and stored in a deep freezer at −80°C awaiting cellular transplantation. Confirmation of Characteristics as LECs Expressions of AFP, albumin, and cytokeratin-19 were detected using Western blotting detection kits (Amersham, Buckinghamshire, UK) according to the manufacturer's protocol. Antibodies, goat polyclonal IgG anti-human AFP, rabbit anti-rat albumin, and anti-human cytokeratin-19 antibody (mouse anti-human cytokeratin-19 antibody; DAKO, Glostrup, Denmark) were used for the detection of AFP, albumin, and cytokeratin-19, respectively. After staining the cells with antibodies against AFP (goat polyclonal IgG anti-human AFP) and albumin (rabbit anti-rat albumin; Inter-Cell Technologies, Hopewell, NJ) using the Avidin-Biotin complex method, the immunohistochemical properties of the cultured cells were examined under a phase-contrast microscope. Efficiency on Isolation of LECs Since the gradient-dependent preparation of the cells alone does not isolate the LECs from other contamination, and a culture step was necessary for purification, the efficiency of LEC isolation from the liver after portal vein ligation was determined by the number of colonies after a 1-week culture of isolated cell preparations. The prepared cells were cultured in an atmosphere of 95% O2 and 5% CO2 on type IV collagen-coated dishes at the cellular density of 7.5 × 104 cells/dish for 1 week at 37°C. The total number of colonies formed on the dishes was then counted as representative of the number of isolated LECs from 1 animal. Transplantation of LECs Into the Liver of NARs After thawing the stored LECs, cellular viability was more than 95%, determined by a Trypan blue exclusion test. After adjusting the viable cell densities, LECs of 2.5 × 106 were transplanted to the livers of 6- to 7-week-old NARs (Charles River Japan, Osaka, Japan) through their portal veins. Livers of the NARs were harvested on days 3, 14, 28, and 56 following transplantation and immunostained with antibodies against AFP and albumin. No immunosuppressive agents were administered to the NARs throughout the experiments. Statistical Analysis Statistical analysis was performed using analysis of variance and Bonferoni's post-hoc test. P-values less than .05 were considered significant and all P-values were 2-tailed. Results Increase in the Number of LECs After Portal Vein Ligation Figure 1 illustrates the microscopic appearance of LECs isolated from a Sprague-Dawley rat liver in this study. They are smaller in size than normal hepatocytes, as are their nuclei. Both Western blotting analysis and immunohistochemical staining revealed that they were AFP-positive, albumin-negative, and cytokeratin-19–positive (Fig. 2). These properties did not change after a 1-week culture. In vivo distribution of LECs in liver tissue in portal vein–ligated lobes is illustrated in Fig. 3. LECs were stained with anti-AFP antibody. LECs were distributed mainly in the periportal area, forming a cluster of cells. There were few LECs found around the central veins. Immunostaining with Ki-67 using serial sections following AFP staining revealed that LECs appeared in the liver were all negative for Ki-67, which demonstrated no proliferative activity of LECs in situ. Figure 1Open in figure viewerPowerPoint The microscopic appearance of liver epithelial cells isolated from livers after portal vein ligation (hematoxylin-eosin staining). Cell size is smaller than hepatocytes, and the nuclei were round. Figure 2Open in figure viewerPowerPoint A: Western blots of alpha-fetoprotein (AFP), albumin, and cytokeratin-19 of liver epithelial cells isolated from livers after portal vein ligation showing AFP-positive, albumin-negative, and cytokeratin-19–positive cells. B: Immunohistochemical staining of liver epithelial cells isolated from livers after portal vein ligation, showing AFP-positive, albumin-negative, and cytokeratin-19–positive cells. Figure 3Open in figure viewerPowerPoint Microscopic and immunohistochemical appearance using serial sections of ligated liver lobes 7 days after portal vein ligation (100×). Alpha-fetoprotein (AFP)-positive liver epithelial cells proliferated mainly in the periportal area and AFP-positive cells were negative for Ki67. Figure 4 shows the changes in number of LECs appeared following portal vein ligation. The population of LECs in the normal liver without any intervention was only 0.79 ± 0.32 cells per 1,000 hepatocytes (n = 4). However, after portal vein ligation, significant increases in the population of LECs were noted from day 3 until day 28 following ligation. On day 7, the population of LECs reached 14.7 ± 6.5 cells per 1,000 hepatocytes (n = 4), about 18 times larger than the population before portal vein ligation. On the other hand, there was no increase in the number of LECs in the nonligated lobes. Figure 4Open in figure viewerPowerPoint Changes in the number of liver epithelial cells (LECs) in ligated and nonligated liver lobes after portal vein ligation. After immunostaining of liver tissue with anti-AFP antibody, the number of LECs was counted (as alpha-fetoprotein (AFP)-positive cells). * P < .05 vs. nonligated lobe. Isolation Efficiency of LECs From the Liver After Portal Vein Ligation Figure 5 shows the colonies on the dish after a 1-week culture. Each colony consisted of more than 500 cells. The efficacy of LEC isolation from livers after portal vein ligations was evaluated by counting the total number of colonies formed on cultures (Table 1). The liver cells isolated without portal vein ligation (control) formed only 1.6 ± 1.4 colonies (n = 4). However, the number of colonies increased dramatically when liver cells were isolated from the portal vein–ligated lobes. Like the population of LECs as compared to total liver cells on tissue sections (Fig. 4), the number of colonies formed in livers was largest on day 7 (67.3 ± 13.9, n = 4). That is, 42 times more LECs were isolated from livers with portal vein ligation than from livers without portal vein ligation. On day 14, the number of colonies decreased to 5.2 ± 2.2 (n = 4), although it was still significantly larger than in the controls. Figure 5Open in figure viewerPowerPoint Phase-contrast microscopic appearance of the colonies formed after a 1-week culture of liver epithelial cells (40×). Table 1. The Number of Colonies Formed After 1-Week Culture of Isolated Cells Control 3 Days After Ligation 7 Days After Ligation 14 Days After Ligation Number of colonies 1.6 ± 1.4 24.2 ± 7.9* 67.3 ± 13.9* 5.2 ± 2.2* Mean ± SD; N = 4 at each time point * P < 0.05 vs. control; control: isolated from the liver before portal vein ligation. LEC Transplantation to Analbuminemic Rat Three days after cellular transplanting isolated LECs to NARs, nests of albumin-positive cells were observed in their livers (Fig. 6A) and were distributed mainly in the periportal areas. These transplanted LECs were thought to have differentiated into hepatocytes, and although they were no longer AFP-positive, they were albumin-positive, demonstrating that they had originally been transplanted LECs because natural hepatocytes in NARs do not produce albumin genetically. These albumin-positive cells were consistently observed in livers until 56 days after transplantation, indicating that the LECs from Sprague-Dawley rats were successfully transplanted and survived in recipient livers (Fig. 6B). Figure 6Open in figure viewerPowerPoint A: Immunostaining with anti-albumin antibody in Nagase analbuminemic rat livers 3 days after transplantation of liver epithelial cells isolated from livers after portal vein ligation (200×). Nests of albumin-positive cells were observed in the periportal areas. B: Immunostaining with anti-albumin antibody in Nagase analbuminemic rat livers 56 days after transplantation of liver epithelial cells isolated from livers after portal vein ligation (200×). Albumin positive cells were consistently observed in livers. Discussion We have previously investigated the character of oval cells isolated from the livers of Long-Evans Cinnamon rats and their differentiation into hepatocytes in vivo.13 Different from other oval cells induced by chemical carcinogens, oval cells from Long-Evans Cinnamon rats were AFP-positive, and albumin-negative, although their shape and size were similar to those of other reported oval cells in the literature.13 In this study, we isolated cells from normal livers and from portal vein–ligated liver lobes using the same isolation procedure as in the previous report.13 Although the method for induction was very different, the LECs that appeared and were isolated in this study proved to be identical to the oval cells reported in our previous study, as they demonstrated the same phenotypic features. Coleman et al.18 have also reported that cultured rat epithelial (stem-like) cells (WB-F344), derived from nonparenchymal cells in normal livers, multiplied in vitro and differentiated into hepatocytes. During galactosamine injury, AFP-positive biliary epithelial cells that proliferate in the periportal area are also likely to be precursors of hepatocytes.19 These findings suggest that hepatic progenitor cells can be isolated from normal livers and can also be induced not only by carcinogenic hepatocellular injury, but also by noncarcinogenic injury. In fact, marked LEC proliferation was evident in our model of portal venous blood flow interception to the liver. Therefore, these results provided us with a new strategy for increasing the number of LECs in liver tissue without using carcinogens. Although the precise mechanism is not clear, it seems directly attributable to ischemic liver injury. Of course, it is also well known that limiting portal venous blood flow gives rise to a loss of hepatocyte-stimulating factors and often results in liver atrophy.20 The decreased stimulation of hepatocytes by the loss of these factors may therefore promote the appearance of LECs. In addition, it is believed that the proliferation of hepatic progenitor cells is triggered by hepatocellular injury, which resulted in the inability of hepatocyte to respond to growth stimuli.21 Based on the results with Ki-67 staining, LECs in the ligated lobe showed no proliferative activities in situ. On the other hand, these cells began to proliferate vigorously to form colonies when they were put into an in vitro environment of a cell culture. This observation was interesting to discern the significance of LECs appeared under this condition. It is still possible that hepatic progenitor cells could appear in the nonligated lobe of the liver as well, but they might disappear very soon because they could promptly differentiate into mature hepatocytes in the presence of portal blood flow. Under microscopic observation, it was observed that the predominant location of LEC proliferation was in the periportal area and the LECs appeared to form in cell clusters. With carcinogenic stimulation, oval cells also appeared and proliferated in the periportal area, extending into the hepatic parenchyma.22 From their investigation of phenotypes, some researchers have suggested the possibility that oval cells derive from cells originating in the bone marrow.23, 24 However, the origin of LECs proliferating in livers after portal vein ligation could not be determined in this study. The method for evaluating efficiency when isolating LECs was complex because the final step in LEC purification included cell cultures. The reason cell cultures were necessary at this final step was because immediately after isolation, cells could be contaminated with a considerable number of nonparenchymal cells and macrophages in the liver. Accordingly, because evaluating isolation efficiency was an objective of this study, it was not feasible to directly compare the isolated cell numbers from the unit weight of the liver between groups. However, since these cells fail to proliferate when cultured, unless growth factors like transforming growth factor-alpha are added,25 the cells forming colonies after a 1-week culture are LECs with self-replication ability. Actually, viable cells, which survived in the culture medium without growth factors, formed colonies, complete with immunohistochemical characters that were AFP-positive and albumin-negative. Therefore, assessment from the number of formed colonies was a rational and practical method to show the isolation efficiency of LECs. Our results determined that the isolation of LECs from ligated-liver lobes after portal vein ligation yielded 42 times more LECs than were found in the normal livers. The difference in the number of LECs between immunohistochemistry and actual isolation may be due to the efficiency of cell separation during the isolation procedure. At any rate, the liver after portal vein ligation is very suitable for isolating LECs, and the best timing for the isolation is approximately 1 week after portal vein ligation. In addition, the result that the time course of isolation efficiency was in accordance with the increase in AFP-positive and albumin-negative cells appearing in the liver indirectly verified that LECs isolated from the liver after portal vein ligation were the same cells as those identified as AFP-positive and albumin-negative cells by immunohistochemistry. The next step of our study was to demonstrate whether these isolated cells could differentiate into hepatocytes. To demonstrate this, a very simple system using NARs was used, as we reported previously.13 The results showed that transplanted LECs transformed into albumin-producing cells, losing their AFP producing ability. They survived in livers for 8 weeks after transplantation. These results illustrate that LECs isolated from livers after portal vein ligation were able to differentiate into hepatocytes in vivo. Another study is in progress concerning long-term follow-up and precise functions of transplanted LECs in this model. In conclusion, LECs in liver tissue markedly increased in number after portal vein ligation, and could be efficiently isolated. They had a self-replicable ability and the capacity to differentiate into albumin-producing hepatocytes. Although further study is necessary, patients whose hepatic lobes undergo portal vein embolization prior to hepatectomy may be good candidates for this source of hepatic progenitor cells. 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