Contribution of extrahepatic small cells resembling small hepatocyte-like progenitor cells to liver mass maintenance in transplantation model of retrorsine-pretreated liver
© Maeda et al.; licensee Springer. 2013
Received: 29 June 2013
Accepted: 27 August 2013
Published: 8 September 2013
Retrorsine selectively inhibits hepatocyte proliferation and following liver injury evokes small hepatocyte-like progenitor cells. The aim of this study is to find out whether endogenous extrahepatic cells contribute to small hepatocyte-like progenitor cells after retrorsine treatment.
Wild-type Lewis rat liver exposed to retrorsine was transplanted into GFP transgenic Lewis rat. GFP positive, albumin-producing polygonal cells were expected as reciepient-derived hepatocyte-like cells.
Four weeks after transplantation of 50% volume of retrorsine-pretreated liver, the rate of GFP positive hepatocyte-like cells was 0.02365%. Majority of these cells resided as single cells and their cell size was significantly larger than that of normal hepatocytes (mean cell size; 799.4 um2 vs. 451.3 um2, p<0.0001). At eight weeks, clusters of GFP positive small-size albumin-producing cells appeared and occupied 0.00759% of hepatocytes. The morphology of these cells was similar to that of small hepatocyte-like progenitor cells, 12.5% of them were Ki67 positive, majority of them were negative for CYP1A2 staining, and some clusters contained larger cells indicating further maturation.
Endogenous extrahepatic cells can form a cluster of small cells resembling small hepatocyte-like progenitor cells in a transplanted retrorsine-pretreated liver. The contribution of extrahepatic cells to liver mass maintenance is quite low and its importance is unclear.
KeywordsSHPC Liver transplantation Retrorsine GFP
Under normal condition, liver regeneration is completed through mitosis of mature hepatocytes. When proliferation of hepatocytes is selectively inhibited by retrorsine treatment, albumin-producing small cells termed small hepatocyte-like progenitor cells (SHPCs) arise in response to liver injuries (Gordon et al. 2000a, bVig et al. 2006Serra et al. 2012). SHPCs form a nodule without a capsule as a result of expansive proliferation, and restore the liver mass. In terms of the cellular origin of SHPCs, several studies did not conclusively determine whether SHPCs were originated from hepatic oval cells (Vig et al. 2006Chen et al. 2013) or mature/maturing hepatocytes (Gordon et al. 2000a, bIchinohe et al. 2012). However, the involvement of bone marrow cells to the formation of nodule of SHPCs is considered less likely (Vig et al. 2006).
In order to study the contribution of bone marrow (BM) stem cells to hepatocytes, BM transplantation models have been often used. However, there is a criticism that procedure-related cell injuries might alter the kinetics of stem cells. In the current study, therefore, we used liver transplantation (LT) model (Tomiyama et al. 2007) instead of bone marrow transplantation model to study the contribution of extrahepatic cells to hepatocyte in a retrorsine pretreated liver. As a result, a cluster of small cells closely resembling SHPCs was identified in the retrorsine-pretreated liver.
Green fluorescence protein (GFP) transgenic Lewis rats were obtained from the national Institute of health (NIH)-funded Rat Resource and Research Center, University of Missouri, Columbia, MO. Male wild type Lewis rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Animals were maintained in the specific pathogen-free facility of the Johns Hopkins Medical Institutions and were cared for according to NIH guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee. Male rats weighting 200-225 g (approximately 8-10 weeks old) received intraperitoneal administration of retrorsine. Male rats weighting 230-280 g were used for liver transplantation.
Liver transplantation (LT)
In order to minimize variation, H. Maeda performed LT exclusively. LT was performed using cuff technique with minor modifications (Kamada and Calne 1979Tomiyama et al. 2007). A donor liver was perfused with cold saline containing 20 units of heparin. For transplantation of a reduced size (50%) liver, left lobe, the left side of right lobe and caudate lobe were resected immediately before saline perfusion. The duration of anhepatic phase, from clamp of portal vein to unclamping of infrahepatic inferior vena cava, was between 15 and 19 minutes. After surgery, all rats were kept individually for at least one month. In the case of death or severe jaundice judged by the color of urine and palm, the rats were excluded from the experiment and additional transplantations were performed to replenish the lost. Severe jaundice was mainly caused by bilestone in common bile duct.
Experimental design and retrorsine pretreatment
Definition of hepatocyte-like cell of extrahepatic cell origin
A hepatocyte-like cell derived from extrahepatic sources was defined as a large polygonal cell which resides in a hepatic cord, expresses GFP in both nucleus and cytoplasm identical to a hepatocyte in GFP transgenic rat (Additional file 1: Figure S1) and exhibits positive staining with anti-albumin antibody (Tomiyama et al. 2007).
Sample preparation and calculation of the contribution rate
One or two month after transplantation, the rats were sacrificed by exsanguination under general anesthesia. The liver was slowly perfused with 50 ml PBS via portal vein, and followed by 50 ml 2% paraformalin. Excised liver was cut into small pieces (5-10 cubic mm) and further fixed with 2% paraformalin for one hour at room temperature in dark. Liver tissues were then incubated in 30% sucrose overnight at four degrees. Two to four pieces of liver samples were randomly embedded in O.C.T. Compound (Sakura Finetek USA, Torrance, CA) and stored in a -80 degree freezer.
To quantitatively analyze the recipient-derived hepatocyte-like cells, pictures of the tissue sections were taken with reference (1 square cm) and then areas of the sections were measured by using Adobe Photoshop CS3 (Adobe systems, San Jose, CA). Then, the number of hepatocytes within 0.16 mm2 from 10 microscopic random fields was examined. Subsequently, 144 cells in the liver without retrorsine treatment and 109 cells in retrorsine-pretreated liver were counted on average. After screening the whole specimen, the rate of extrahepatic cell contribution was calculated. In order to avoid counting the same cell redundantly, the sections were prepared at least 36 μm apart from each other.
Six μm tissue sections were incubated with 1% SDS (detergent) in PBS for 12 minutes. For albumin staining, sections were incubated with sheep anti-rat albumin antibody (1:800 dilution, BETHYL laboratory Inc, Montgomery, TX) for 30 min, followed by Cy3-conjugated donkey anti-sheep IgG antibody (1:400 dilution, Jackson immunoreserch laboratories, west Grove, PA) for 30 min. For Ki67 staining, sections were incubated with mouse anti-human Ki67 antibody (1:50 dilution, BD Pharmingen, San Jose, CA) for 60 min, followed by Cy3-conjugated donkey anti-mouse IgG antibody (1:100 dilution, Jackson immunoreserch laboratories) for 30 min. For CYP1A2 staining, sections were incubated with mouse anti-rat CYP1A2 (1:100 dilution, Abcam, Cambridge, MA) antibody for 60 min, followed by Cy3-conjugated donkey anti-mouse IgG antibody for 30 min. Nuclear staining was performed with DAPI (DAKO, Cambridgeshire, UK) when necessary. Serum blocking was performed with 10% donkey serum.
The tissue sections were incubated with 1% SDS in PBS for 12 minutes. Then, they were incubated in 1% H2O2 in PBST for 25 min. After avidin, biotin block (Avidin blocking system; DAKO), sections were incubation with biotin conjugated anti-GFP antibody (Abcam) for one hour. Tissue sections were then incubated with AB complex (VECTASTAIN ABC kit; Vector, Burlingame, CA) for 30 min according to the manufacture instruction and reacted with DAB (Liquid DAB+ Substrate Chromogen System; DAKO). Counterstaining was performed by using Haematoxylin for 20 seconds. After each step, sections were adequately rinsed with PBST. When the same specimen was stained for GFP following albumin observation, incubation with 1% SDS was not performed again.
To evaluate proliferation of oval cells after LT, OV-6 expression was studied on immunohistochemistry staining. Sections were incubated in 1% H2O2 in PBST for 25 min. Then, non-specific staining was reduced by incubation with 5% donkey serum for 10 min. After avidin biotin block, sections were incubated with anti-OV-6 antibody (1:50 dilution, Santa Cruz Biotechnology, Dallas, TX) for one hour followed by biotin-conjugated anti-mouse antibody (1:100 dilution, Jackson immuneresearch laboratories) for 30 min. The signal was amplified with AB complex and detected with DAB.
Statistical analysis and graphic depiction was performed by using KaleidaGraph version 4.0 (synergy software, Reading, PA) or Microsoft Office Excel (Microsoft, Redmond, WA). A p- value of less than 0.05 was considered statistically significant. The method of analysis is described individually.
Mortality and exclusion of rats
Most recipients survived (19 out of 20, 95%) irrespective of whether rats received retorsine-pretreated or untreated livers. One recipient rat died and one rat was excluded due to severe jaundice. Additional LT was uneventfully performed.
GFP positive hepatocyte-like cells in livers without retrorsine treatment (Group 1 & 2)
Rate of extrahepatic cell contribution to hepatocytes after retrorsine-pretreated liver transplantation
Screened hepatocytes (cells)
GFP positive hepatocyte-like cells (%)
Cluster of cells(%)
Group 1 (Whole LT) 8 weeks
Group 2 (50% LT) 8 weeks
Group 3 (whole LT & retrorsine) 4 weeks
Group 3 (whole LT & retrorsine) 8 weeks
Group 4 (50% LT & retrorsine) 4 weeks
Group 4 (50% LT & retrorsine) 8 weeks
Histological changes after transplantation of retrorsine-pretreated liver
GFP positive hepatocyte-like cells and cluster of small cells in livers with retrorsine-pretreatment (Group 3 & 4)
The rate of GFP positive hepatocyte-like cells in retrorsine treated small livers (Group 4) was 0.02365% at four weeks. Majority of GFP positive hepatocyte-like cells were abnormally large as observed in Group 3. However, the rate GFP positive hepatocyte-like cells decreased at eight weeks (0.01056% vs. 0.02365%, p<0.05, Mann-Whitney U test) as the size of nodule of GFP negative SHPCs enlarged. Clusters of GFP positive cells were also identified in tissue sections at 8 weeks in Group 4, which rate was also very low (Additional file 1: Table S1).
Size of GFP positive hepatocyte-like cells and SHPCs-like cells
After transplantation, the size of GFP negative hepatocytes was widely distributed and slightly larger than that of hepatocytes in nontransplanted normal livers (median; 463.1 μm2 vs. 451.3 μm2, p=0.049). GFP positive hepatocyte-like cells in the livers of Group 1 and 4 were significantly larger than GFP negative hepatocytes in Group 1 and control (median; 709.5 μm2 and 726.3 μm2 versus 463.1 μm2 and 451.3 μm2, p<0.0001), although the some of these cells were similar to normal hepatocytes in size. The size of GFP positive SHPCs-like cells was similar to GFP negative SHPCs (351.6 and 390.3 μm2, respectively), and both of these cells were smaller than GFP negative hepatocytes in Group 1 (p<0.01).
Proliferation of GFP positive SHPC-like cells
Rate of Ki67 positive hepatocyte
GFP (+)hepatocyte-like cells
GFP (-) SHPCs
GFP (+)SHPCs-like cells
Oval cell proliferation after LT
Hepatic injury causes occurrence of SHPCs in retrorsine-treated liver, and they gradually restore the loss of hepatocytes or replace the injured hepatocytes (Serra et al. 2012Gordon et al. 2000a, bVig et al. 2006). Although SHPCs have some of phenotype makers in common with oval cells or mature hepatocytes (Gordon et al. 2000a, b), the most intrinsic features of SHPCs are their small size, albumin production and formation of nodules. SHPCs also lack in expression of some CYP isozymes at immature stage. Because retrorsine is metabolized into active forms in hepatic microsomes and then selectively damages hepatocytes, the lack of CYP expression might be the mechanism of retrorsine resistance of SHPCs (Gordon et al. 2000b). The origin of SHPCs is largely endogenous cell population of the liver and contribution of bone marrow cells is considered less likely (Vig et al. 2006). In the current study using LT model, however, we identified the clusters of GFP positive small cells fulfilling these characteristic features of SHPCs for the first time. Also, GFP positive SHPCs-like cells at eight weeks showed high rate of Ki67 positive cells and several size of nucleus, suggesting their ability of proliferation and maturation. Besides, the retrorsine pretreated liver showed clusters of GFP positive hepatocytes at least more than six month after transplantation, suggesting long-term survival in the transplanted liver (data not shown). However, the biological importance of GFP positive SHPCs-like cells is unclear due to the very low rate of occurrence, and a further study is necessary.
Whether the contribution of extrahepatic cells to hepatic parenchymal cells is accomplished through transdifferentiation or cell fusion is also frequently debated. This could not be directly addressed due to the rarity of GFP positive hepatocyte-like cells and therefore the cell sizes were focused in the current study. As a result, the size of GFP positive hepatocyte-like cells was found larger than surrounding GFP negative hepatocytes in the liver of Group 1. Because GFP positive hepatocyte-like cells occasionally resided adjacent to each other or showed Ki67 positive staining, some of them are under the process of cell proliferation. However, the rate of Ki67 positive cells among GFP positive hepatocyte-like cells is not higher than that among GFP negative hepatocytes. Similarly, the GFP positive hepatocyte-like cells in retrorsine-pretreated liver showed abnormally large size and irregular shape. These results may imply that GFP positive hepatocyte-like cell is formed by cell fusion rather than transdifferentiation of extrahepatic cells into hepatocytes. Furthermore, we also performed LT transplantation of opposite direction, from GFP transgenic rat liver to a wild type rat. In this model, no GFP negative megalocytic hepatocyte was found at four weeks (Additional file 1: Figure S3). However, this approach didn’t provide a clue about the pathway of contribution of extrahepatic cells to SHPCs-like cells.
In this experiment, LT model was used instead of BM transplantation in order to find the contribution of extrahepatic cells to hepatocytes. We believe the influence on bone marrow is limited in LT model because damage on BM cells is short periods of congestion in rear part of the body during the clamping of IVC. This may explain why the current study showed GFP positive small cells resembling SHPCs and a previous experiment did not identified similar cell population (Vig et al. 2006). However, LT model also has unique problems. First, it cannot specify the origin of the extrahepatic cells. Being consistent with majority of published data, we found a very few GFP positive hepatocyte-like cells in wild-type livers after BM transplantation from GFP transgenic rats (data not shown). Therefore, BM is one of the most likely sources of hepatocytes in LT models, too. However, other organs possess resident stem progenitor cells, and these cells are potentially mobilized into peripheral circulation and take part in organ repair (Kolonin 2012). Compared to BM, from which BM cells are continuously released into peripheral blood, the contribution of stem cells from other organs under physiological condition should be minimal. However, balance of contribution may change under pathological condition (Kolonin 2012), which study has only just begun. Second, liver transplantation itself causes acute and chronic liver injuries. Abnormalities in bile duct system due to lack of arterial reconstruction (Hori et al. 2012) and the use of stent for bile duct reconstruction also caused intra- and/or extra hepatic cholelithiasis almost in all cases in this study. Probably, these factors caused differences in cell sizes and Ki67 expression of endogenous hepatocytes between control (no transplantation) and LT groups. Chronic liver injury due to cholelithiasis also seems to be the reason of gradual and persistent emergence of SHPCs and continuous oval cell reaction after whole transplantation of retrorsine-pretreated liver.
The limitation of our study is that GFP is the only marker used to detect the extrahepatic cells. Because of the very low rate of extrahepatic cell contribution, detection of X or Y chromosome as a marker of extrahepatic cells after sex-mismatch transplantation seems difficult. Also, due to the usage of paraformalin for better preservation of GFP signals, further characterization of GFP positive SHPCs-like cells by detecting the weaker cell markers was difficult. Despite limitations, the current study found that extrahepatic cells can form a cluster of small cells which closely resemble SHPCs in retrorsine-pretreated transplanted rat liver. The rate of contribution is quite low and further study is necessary to illuminate its biological importance.
Small hepatocyte-like progenitor cells
Green fluorescence protein.
HM was supported partly by Uehara Memorial Foundation.
The Uehara Memorial Foundation to HM.
- Chen YH, Chang MH, Chien CS, Wu SH, Yu CH, Chen HL: Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers. Hepatology 2013, 57: 1215-1224. 10.1002/hep.26104View ArticleGoogle Scholar
- Gordon GJ, Coleman WB, Hixson DC, Grisham JW: Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol 2000, 156: 607-619. 10.1016/S0002-9440(10)64765-7View ArticleGoogle Scholar
- Gordon GJ, Coleman WB, Grisham JW: Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats. Am J Pathol 2000, 157: 771-786. 10.1016/S0002-9440(10)64591-9View ArticleGoogle Scholar
- Hori T, Gardner LB, Chen F, Baine AM, Hata T, Uemoto S, Nguyen JH: Impact of hepatic arterial reconstruction on orthotopic liver transplantation in the rat. J Invest Surg 2012, 25: 242-252. 10.3109/08941939.2011.636476View ArticleGoogle Scholar
- Ichinohe N, Kon J, Sasaki K, Nakamura Y, Ooe H, Tanimizu N, Mitaka T: Growth ability and repopulation efficiency of transplanted hepatic stem cells, progenitor cells, and mature hepatocytes in retrorsine-treated rat livers. Cell Transplant 2012, 21: 11-22.View ArticleGoogle Scholar
- Kamada N, Calne RY: Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 1979, 28: 47-50. 10.1097/00007890-197907000-00011View ArticleGoogle Scholar
- Kolonin MG: Progenitor cell mobilization from extramedullary organs. Methods Mol Biol 2012, 904: 243-252.Google Scholar
- Serra MP, Marongiu F, Sini M, Laconi E: Hepatocyte senescence in vivo following preconditioning for liver repopulation. Hepatology 2012, 56: 760-768. 10.1002/hep.25698View ArticleGoogle Scholar
- Tomiyama K, Miyazaki M, Nukui M, Takaishi M, Nakao A, Shimizu N, Huh NH: Limited contribution of cells of intact extrahepatic tissue origin to hepatocyte regeneration in transplanted rat liver. Transplantation 2007, 83: 624-630. 10.1097/01.tp.0000253942.16061.d9View ArticleGoogle Scholar
- Vig P, Russo FP, Edwards RJ, Tadrous PJ, Wright NA, Thomas HC, Alison MR, Forbes SJ: The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice. Hepatology 2006, 43: 316-324. 10.1002/hep.21018View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.