Isolation and Characterization of Bipotent Liver Progenitor Cells from Adult Mou - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Wen-Lin Lia, Juan Sua, Yu-Cheng Yaoa, Xin-Rong Taoa, Yong-Bi Yana, Hong-Yu Yub, Xin-Min Wanga, Jian-Xiu Lia, Yong-Ji Yanga, Joseph T.Y. Lauc, Yi-Ping Hua作者单位：a Department of Cell Biology, Second Military Medical University, Shanghai, Peoples Republic of China;b Department of Pathology, Changzheng Hospital, Shanghai, Peoples Republic of China;c Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York, USA
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      【摘要】% l: N9 B& f! S0 ~- U3 m
   Liver progenitor cells have drawn a great deal of attention both for their therapeutic potential and for their usefulness in exploring the molecular events surrounding liver development and regeneration. Despite the intensive studies on liver progenitors from rats, equivalent progenitor cells derived from mice are relatively rare. We used retrosine treatment followed by partial hepatectomy to elicit liver progenitors in mice. From these animals showing prominent ductular reactions, mouse-derived liver progenitor cell lines (LEPCs) were isolated by single-cell cloning. Phenotypic and lineage profiling of the LEPC clones were performed using immunochemistry, reverse transcription¨Cpolymerase chain reaction, and a dual-color system comprising the reporter EGFP under the control of the cytokeratin 19 promoter and the DsRed reporter under the control of the albumin promoter. LEPCs expressed liver progenitor cell markers. LEPCs also expressed some markers shared by bone marrow-derived hematopoietic stem cells c-Kit and Thy-1 but not CD34 and CD45. When cultured as aggregates in Matrigel, LEPCs differentiated into hepatocyte upon treatment with 50 ng/ml epithelial growth factor or differentiated into biliary lineage cells upon treatment with 20 ng/ml hepatocyte growth factor. In the presence of 2% dimethyl sulfoxide and 2% Matrigel, LEPCs acquired predominantly bile lineage phenotypes, with occasional patches of cells exhibiting hepatocyte phenotypes. Upon transplantation into CCl4-injured-liver, LEPCs engrafted into liver parenchyma and differentiated into hepatocytes. Considering the amenability of the mouse to genetic manipulation, these mouse-derived LEPCs may be useful tools as in vitro models to study molecular events in liver development and regeneration and can shed light in studying the therapy potential of liver stem cells.
      【关键词】 Liver progenitor cell Stem cell Differentiation Liver regeneration' t9 \) s, `  I2 g
   INTRODUCTION" t$ {5 ]+ Q$ ]9 b3 i
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Identification of multipotent stem cells in mammalian tissues is of great interest not only because of the therapeutic possibilities but also for better understanding developmental processes and tissue homeostasis .
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A model involving an initial administration of the DNA-binding alkaloid retrosine (Rs) to block native hepatocyte proliferation and subsequent partial hepatectomy (PH) serving as mitogenic signal has been used to study liver regeneration  did not find SHPCs during liver regeneration, but the proliferation of small amounts of bile duct-associated oval cells was observed.
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Although rat adult liver¨Cderived progenitors are intensively investigated, equivalent progenitor lines from the mouse are relatively rare  to elicit liver regeneration. Whereas no SHPCs were observed, there were obvious oval cell¨Clike cell clusters near the portal tracts. Most of the medium ductular reaction appeared 10 to 14 days after PH, and there was no ductular reaction without Rs administration. Culturing the cells from the regenerating livers exhibiting ductular reactions resulted in the establishment of oval cell¨Clike liver progenitors, which we named liver epithelial progenitor cells (LEPCs). Like the rat oval cells, the mouse LEPCs show the capacity for bipotent differentiation under different conditions, and this trait can be useful for elucidation of the molecular signals required for hepatic cell lineage plasticity. Moreover, when transplanted into acute injured liver induced by CCl4, LEPCs engrafted into the liver parenchyma and differentiated into hepatocytes., ^: {& q0 i0 [+ v# p/ g
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MATERIALS AND METHODS

Animals

Normal male C57BL/6 mice, 8 to 10 weeks of age, weighing 20 to 30 g, were obtained from the Transgenic Animal Research Center, Second Military Medical University (specific pathogen-free grade). The Rs (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) coupled with PH administration protocol was as reported . Surviving mice in the Rs/PH (n = 35) and control/PH (n = 27) groups were killed, and livers were harvested at 2, 4, 6, 8, 10, 14, 18, 24, and 30 days after PH (n = 3 per time point) for morphological studies. All animal procedures were performed in accordance with institutional guidelines.
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Isolation, Culture, and Clone of LEPC Line; p: c' I) ]8 K7 d" t

Cells were isolated from mice 14 days after Rs/PH using the two-step collagenase perfusion method . After perfusion, the livers were excised and pelleted into small pieces in 0.1% collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ, http://www.worthington-biochem.com) and 0.05% DNAase I (Worthington) solution in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) and then incubated at 37¡ãC for 30 minutes. Primary liver cell suspensions were centrifuged two times at 50g for 1 minute. Each time, the pellet was discarded, and then the supernatant was centrifuged two times at 50g for 2 minutes. Cell pellet was suspended in DMEM with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, http://www.hyclone.com). Cell viability was determined by trypan blue exclusion assay and plated onto 60-mm uncoated dishes (Greiner Bio-One, Fricken-hausen, Germany, http://www.gbo.com) at a density of 1,000 viable cells/mm2. Two hours later, the nonadherent cells were discarded by medium change. The cells were maintained in culture medium: DMEM HG, 1% penicillin/streptomycin (Gibco-BRL), 10 µg/ml insulin (Sigma-Aldrich), 1 mM Glutamax (Gibco-BRL), and 10% FBS at 37¡ãC with 5% CO2. The culture medium was half changed every 2 days. Cells were examined with a light microscopy every other day. The fibroblasts were selectively detached from culture by repeated differential trypsinization with a solution of 0.05% trypsin in 0.02% EDTA at room temperature and scraping by cell scraper (Sarstedt, Newton, NC, http://www.sarstedt.com). The purified epithelial populations were named LEPCs and passaged every 3 days. The tenth-passage LEPCs were serially diluted and re-plated onto three U-bottom 96-well plastic tissue culture plates (one-half cell in 100 µl culture medium per well). Cultured wells containing only one cell were marked (total, 85 wells) and observed every 3 days under phase-contrast microscopy. Upon reaching confluency, the clones from each well were dissociated and plated in six-well plastic tissue culture plates. Eventually, 10 clones were selected and named numerically as cloned LEPCs 1 through 10.
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Immunocytochemistry and Fluorescence-Activated Cell Sorter Analysis" I/ x- ~4 [2 e2 y: L6 a9 e

The LEPCs 2, 4, 8, and 10, growing in cover glass, were fixed in cold 95% ethanol for 30 minutes. Rabbit polyclonal anti-albumin (ALB) (1:100) (ICN/Cappel, Aurora, OH, http://www.mpbio.com), TROMA-3 rat monoclonal anti-CK19 (1:100) (kindly provided by R. Kemler, MPI, Germany), rat monoclonal anti-c-Kit (1:500) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit polyclonal Thy-1 (1:500), and CD34 (1:250) (Santa Cruz Biotechnology) antibodies were used as primary antibodies. The cells were incubated with the first antibody for 16 hours at 4¡ãC and then stained with the Dako Envision detection system (Dako, Carpinteria, CA, http://www.dakousa.com). Liver tissues were fixed in 4% phosphate-buffered paraformaldehyde overnight at 4¡ãC and embedded in OCT compound. Liver cryostat sections 10 µm in size were incubated with rabbit polyclonal -1 anti-trypsin antibody (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) for 16 hours at 4¡ãC and then stained with Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com).; V+ ~/ t% i( f8 J" u) c

Indirect flow cytometry (fluorescence-activated cell sorter ) staining for CK19 and c-Kit was carried out on ice, making use of the TROMA-3 and rat monoclonal anti-c-Kit antibodies and Alexa Fluor 488 goat anti-rat IgG as second antibody. Thy1 staining was carried out using the FITC-conjugated rat monoclonal anti-Thy-1 antibody (Santa Cruz). All samples were analyzed on a FACStar flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Reverse Transcription¨CPolymerase Chain Reaction Analysis( }2 W# X! D/ o
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Total cellular RNA was extracted from LEPCs 1 through 10 by Trizol reagents according to standard protocols. Reverse transcription (RT) was performed with 2 µg RNA, random nonamers (TaKaRa, Dalian, LiaoNing, China, http://www.takara.com.cn), and murine Moloney leukemia virus RT (Promega, Madison, WI, http://www.promega.com) according to the manufacturer¡¯s manual. The polymerase chain reaction (PCR) conditions were 95¡ãC for 5 minutes, 94¡ãC for 30 seconds, annealing temperature for 30 seconds, and 72¡ãC for 60 seconds, 25 to 35 cycles, and then 72¡ãC for 10 minutes. Forward and reverse primers used for specific amplification can be found in the references for the following molecules: ALB, GS (glutamine synthase), tryptophan-2, 3-dioxygenase, CK19, CK18, CK8, c-Met, c-Kit, Thy-1, and CD34 .% V8 F* t# x4 l* ~( |8 `
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Vector Construction and LEPC Transfection/ t( {7 U3 a) O- X5 D2 t) r. ~
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To construct pAlb-DsRed, the 2.3-kb ALB enhancer/promoter fragment was recovered from plasmid pGEMAlbSVPA (a kind gift from Professor Derek LeRoith, NIH) and sub-cloned into pDsRed1 1 (Clontech, Palo Alto, CA, http://www.clontech.com). To construct pCK19-EGFP, the CK19 promoter 5' UTR (accession no. AF237661  was amplified by PCR from mouse tail genomic DNA by primers 5'-GTTCCTTTCTAAGACCCA-3' and 5'-TAGTG-GTTGTAATCTCGG-3'. The authenticity of the 2-kb PCR product was confirmed by sequence analysis. A Vsp1/Xba1 fragment of the PCR product using primers 5'-AAATTAAT-TCTAAGACCCACC-3' and 5'-GATAGTCTAGAGAAGT-CATGATG-3', containing CK19 promoter, was ligated with Vsp1/Nhe1 CMV promoter-removing fragment of pEGFP C1 vector (Clontech). After introducing pCK19-EGFP into HeLa and NIH-3T3 cells, EGFP expression was detected in HeLa but not in NIH-3T3 cells. To construct pCMV-(CK19/EGFP), full-length CK19 cDNA was amplified by RT-PCR from the total RNA of LEPC 2 by primers 5'-TCTCCCTCCTCAT-CATGACTTCC-3' and 5'-TCGCTGGTAGCTCAGATGGC-3'. The validity of the 1.2-kb fragment was confirmed by sequence. Xho1/EcoR1 PCR fragment of CK19 cDNA (by primers 5'-TCTCTCGAGTCATCATGACTTCC-3' and 5'-TCGGAATTCGCTCAGATGGC-3') was ligated into Xho1/EcoR1-digested pEGFP C1 vector. In this configuration, CMV immediate early promoter controlled the expression of (CK19/EGFP) fused protein. The vectors were introduced into LEPCs 2, 4, 8, and 10 by electroporation. After 2 weeks of selection by G418 (800 µg/ml), the individual G418-resistant clones were picked up and subcultured.4 }$ g' Z/ [% s7 M! B7 `( T# J
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In Vitro Differentiation
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One milliliter of Matrigel (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com) was suspended in 50 ml DMEM HG medium supplement with 10% FBS and frozen at ¨C20¡ãC overnight and then thawed at 4¡ãC overnight to get homogeneous 2% Matrigel medium. A total of 3 x 105 cloned LEPCs 2, 4, 8, and 10 were suspended in 3 ml 2% Matrigel medium supplement with 2% dimethylsulfoxide (DMSO) and plated onto 60-mm dishes. The total RNA was extracted after 10 days of induction.' A; I. B$ |7 h5 m6 M
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Matrigel 0.5 ml mixed with 0.5 ml serum-free DMEM HG medium supplement with epithelial growth factor (EGF) (Sigma-Aldrich) at a final concentration of 50 ng/ml or hepatocyte growth factor (HGF) (Peprotech, London, http://www.peprotechec.com) at a final concentration of 20 ng/ml was added into the wells of six-well plates. After incubation at 37¡ãC for 2 hours to allow to gel, 105 cloned LEPCs 2, 4, 8, and 10 were plated above the gel carefully. The total RNA was extracted after 5 days of incubation.
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LEPC Transplantation

To follow the differentiation of LEPCs in vivo, pAlb-DsRed or pCK19-EGFP-transfected LEPC 2 (1 x 106) was suspended in DMEM and injected into the spleens of C57BL/6 mice as described previously (n = 10, 8 weeks old) . Mice were killed 1 and 6 months after cell implantation. Recipient livers were fixed in 4% phosphate-buffered paraformaldehyde overnight at 4¡ãC and embedded in OCT compound. Liver cryostat sections in 10 µm were observed under a fluorescence microscope to detect the transplanted cells. Quantification of DsRed-positive cell participation in liver was performed in cryostat sections of left lobes (10 random x 100 visual fields were selected to get mean value for every mouse) by using PhotoShop 6.0 (Adobe Systems). The percentage of cell participation was calculated from the number of pixels in each visual field of liver cryostat sections compared with the number of pixels of DsRed-positive fields.
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RESULTS: j7 h, o& `! p% B

Oval-Like Progenitor Cells During the Regeneration of Mouse Liver After Rs Treatment Coupled with 2/3 PH, o; C6 _2 m5 a- x

After Rs/PH treatment, 30% of Rs-treated mice exhibited distinct ductular reaction 10 to 14 days after PH. Oval cell¨Clike cells appeared in some but not all Rs-treated mice by approximately 10 to 14 days after PH. Small cells with round nuclei and scant cytoplasm that morphologically resemble classic oval cells were located in the vicinity of portal tracts, and this response was absent in control mice that were subjected to PH but without prior Rs administration. These cells proliferated between the larger hepatocytes and radiated from portal tracts forming primitive ductular structures with poorly defined lumen (Figs. 1A, 1B). Significantly, no morphologically distinct SPHC clusters were observed in mice after Rs/PH treatment.) N8 ^" Y" u( m- U
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Figure 1. Establishment of liver epithelial progenitor cell (LEPC) lines from retrosine (Rs)/partial hepatectomy (PH)-treated mice. The emergence of oval cell¨Clike cells in Rs/PH-treated mice 14-day after PH (A) (hematoxylin and eosin staining). (B): Magnification of the outlined area in (A). Small oval cell¨Clike cells (arrows) were observed in Rs/PH-treated mice. Arrowheads indicated the primitive ductular structures with poorly defined lumen. (C¨CI): Phase-contrast photographs of LEPCs isolated from Rs-treated mouse. Five days after the primary culture, some epithelial clones were observed (C). Epithelial clones proliferated in the culture (D). LEPCs were established after removing fibroblast; the population was heterogeneous in morphology (E). By single-cell cloning, LEPCs 1 through 10 were selected and expanded as homogeneous epithelial clones; LEPCs 2, 4, 8, and 10 are shown in F, G, H, and I, respectively. Relative magnification: A, C-I, x200; B, x400.# s8 {* X8 i9 S, U% o; m) A

Establishment of LEPC Lines from Rs/PH-Treated Mice

LEPCs were derived from regenerative liver showing distinct ductular reactions 14 days after Rs/PH. After depletion of the nonadherent cells, a primary culture was established. Five days after plating, some clusters containing small epithelial cells were observed (Fig. 1C). No similar epithelial cell clusters appeared in the primary culture from control mice subjected to two-thirds PH without the retrosine pretreatment. The epithelial cells pro-liferated slowly but continuously (Fig. 1D). By repeated differential trypsinization and passage, fibroblasts gradually diminished from culture. Epithelial cell population was established in cobblestone fashion with high nuclear-to-cytoplasm ratios (Fig. 1E). The cell lines, expressing liver progenitor markers and showing biopotency (described below), were named LEPCs. LEPCs have normal 38 XY karyotypes. From the FACS analysis, the DNA content (4N) values of  phases in the cell cycle were 87.72%, 2.54%, and 9.74%. LEPC cells (passage 30) did not develop any tumor in nude mice after 4 months of observation. By single-cell cloning, a total of 28 clonal LEPCs were obtained from the tenth passage of LEPCs according to slight differences in clone shape and cell size; 10 clonal LEPCs were selected and expanded. The doubling time of the 10 expanded clonal LEPCs was between 20 and 36 hours. Among these 10 lines, 7 remained diploid in nature, and none of the 10 lines developed into tumors upon inoculation into nude mice. Among these 10 clonal lines, the DNA content of LEPCs 4, 5, and 9 increased. On the other hand, LEPCs lines 2, 4, 8, and 10 remained homogeneous epithelial cells (Figs. 1F-1I) and have been stably cultured in vitro for more than 18 months (70 passages) without DNA content change and proliferation decrease. LEPCs 2, 4, 8, and 10 were further studied as described below.8 h" D( G5 V9 B$ o6 L! p
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LEPCs Express Oval Cell/Bile Duct Lineage Markers and Liver-Enriched Transcription Factors
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Antigens traditionally associated with hematopoietic stem cells, including Thy-1, c-Kit, and CD34, have been reported to be expressed by oval cells . By immunochemistry method, we show that LEPCs 2, 4, 8, and 10 expressed the hematopoietic stem cell markers c-Kit and Thy-1 but not CD34 and CD45. Consistent with LEPCs being oval cell¨Clike, these LEPCs also express CK19, the cytokeratin protein serving as a marker for biliary lineage (Figs. 2A¨C2C). pCK19-EGFP-transfected LEPCs showed homogeneous green fluorescence (Fig. 2F), which further supported the CK19 expression in LEPCs. It is noteworthy that the intracellular distribution of CK19 was nonuniform, as revealed by staining using anti-CK19 (Fig. 2A), and the nonuniform distribution could be reproduced by the transfected CK19/EGFP fusion protein expressed in LEPCs 2, 4, 8, and 10 (Figs. 2D, 2E).
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Figure 2. Immunocytochemical detection oval cell markers CK19 (A), c-Kit (B), and Thy-1 (C) in liver epithelial progenitor cell (LEPC) 2. Arrowheads indicate the uneven distribution of CK19 staining. The pCMV (CK19/EGFP)-transfected LEPC 2 showed polarity green fluorescence distribution (D). (E): Overlay image of (D) and corresponding phase-contrast photograph. The pCK19-EGFP-transfected LEPC 2 clone showed homogeneous green fluorescence (F). Fluorescence-activated cell sorter analysis showed LEPC 2 expressed CK19 (96.48%), c-Kit (89.58%), and Thy-1 (43.64%) (G). Relative magnification: A-C, x200; D, E, x400; F, x100.
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Based on FACS analysis, LEPC 2 expressed CK19, c-Kit, and Thy-1 with 97%, 90%, and 44% of the total population positive for these markers, respectively (Fig. 2G). RNA from LEPCs 1 through 10 was analyzed by RT-PCR, and the results are shown in Figure 3. All of the clonal cell lines expressed the cytokeratin proteins CK8, CK18, and CK19, the hepatocyte grow factor receptor c-Met, and liver-enriched transcription factors HNF 1, HNF 1ß, HNF 3, HNF 3ß, and HNF 6. LEPCs 2, 3, 5, and 8 expressed HNF 4 at a low level. In agreement with immunochemistry and FACS analysis, LEPCs expressed oval cell/hematopoietic cell markers c-Kit and Thy-1 but not CD34 and CD45. LEPCs lines expressed hepatocyte marker ALB and bile duct marker GGT in low levels. All cell lines expressed mature hepatocyte molecule GS but did not express liver highly functional gene tryptophan-2,3-dioxygenase.2 K! A0 j/ }- U* @. K. R. w
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Figure 3. Reverse transcription¨Cpolymerase chain reaction (RT-PCR) analysis of liver epithelial progenitor cells (LEPCs) expressing oval cell/bile duct lineage markers and liver-enriched transcription factors. PCR was performed for 30 cycles. Abbreviations: L, 4-week-old mouse liver tissue; HPRT, internal loading control. No signal was detected in control samples (not treated with RT).9 Q' |0 _/ |+ K) h7 f& Q

LEPCs Are Bipotential Liver Progenitor Cells% |4 n% N  b: \3 Z5 @' y% ?) l
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To investigate the plasticity of LEPCs in vitro, we cultured the LEPCs in the extracellular matrix Matrigel in the presence of the general differentiation-inducing agent DMSO or in the presence of cytokines (EGF or HGF). DMSO has been shown to be useful in maintaining hepatocyte differentiation phenotype in vitro . In the basal medium, LEPCs proliferated as small, polygonal cells with a high nuclear/cytoplasmic ratio. LEPCs proliferation was arrested upon exposure to a medium with 2% Matrigel and 2% DMSO for 3 days, along with an increase in cell size and shape lengthening (Fig. 4A).

Figure 4. Phase-contrast photograph of the mixture of pCK19-EGFP and pAlb-DsRed-transfected LEPC 2 clones. Ten days after plating by medium supplement with 2% Matrigel and 2% dimethylsulfoxide, liver epithelial progenitor cells (LEPCs) elongated and proliferation was inhibited (A). The intensity of green fluorescence was significantly enhanced in some cell clusters (B). Ultrastructural observation showed characteristics of bile duct¨Clike cells with basement membrane on one side and microvilli on the opposite side (C, D). (D): Magnification of the outlined area in (C). A small patch of DsRed-positive cells, indicating the Alb expression, was also observed after induction (E¨CG). (F): Magnification of outlined area in (E). (G): Corresponding fluorescence photograph for (E). FACS analysis revealed approximately 0.20%, 1.16%, and 3.12% cells expressing DsRed at 0, 5, and 10 days after treatment. Untransfected LEPC 2 was used as negative control (H). Arrows indicate the basement membrane. Arrowhead indicates the microvilli. Magnification: A, B, E, G, x200; F, x400. Bar = 1 µm.; N7 {$ A( b) a% T$ h
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To assist in monitoring phenotype variation, the reporter plasmids pCK19-EGFP and pAlb-DsRed were constructed to follow differentiation into biliary or hepatocyte lineage, respectively. LEPCs transfected with either pCK19-EGFP or pAlb-DsRed and pooled together in a 1:1 ratio were used. When the pooled LEPCs were plated in the presence of Matrigel and DMSO, the green fluorescence of some cells, resulting from expression of EGFP under the control of the CK19 promoter, enhanced gradually during this time. After 10 days of induction by Matrigel and DMSO, a strong green fluorescence can be observed in many cell clusters (Fig. 4B), suggesting the upregulation of biliary phenotype. Ultrastructurally, the cells displayed bile duct cell¨Clike features with basement membrane on one side and microvilli on the other side (Figs. 4C, 4D). Interestingly, we also observed small patches of cells with red fluorescence, resulting from expression of DsRed controlled by the Alb promoter from the hepatocyte-specific albumin (Figs. 4E¨C4G). DsRed-positive cells exhibited the morphological traits of hepatocytes, which were quite different from the surrounding EGFP-positive cells. Some tiny canals, like bile canaliculus, can be observed between two adjacent cells (Figs. 4E-4G). FACS analysis revealed that approximately 0.2%, 1.2%, and 3.1% of the total population were DsRed-positive at 0, 5, and 10 days after DMSO treatment (Fig. 4H), and the abundance of DsRed-positive cells did not increase significantly after 10 days. Based on the 1:1 ratio of input LEPCs containing pCK19-EGFP or pAlb-DsRed, we extrapolate the frequency of LEPC differentiation into hepatocyte lineage to be 2.4% and 6.2% after 5- and 10-day exposure to Matrigel and DMSO, respectively. These results suggested the bipotential ability of LEPCs to differentiate into bile duct cells and, much less frequently, to hepatocytes when cultured in Matrigel and DMSO. By RT-PCR analysis, Matrigel and DMSO increased the level of biliary lineage markers such as CK19, aquaporin-1, and GGT, with concomitant decrease in the hepatocyte molecule GS (Fig. 5A). This RT-PCR profile confirmed that LEPCs tended to differentiate into bile duct cells in the presence of Matrigel and DMSO. However, the induction was not entirely exclusive because the hepatocyte marker albumin was also upregulated (Fig. 5A).) `' o' f' O* n& `4 x- f; }4 ?3 p

Figure 5. Reverse transcription¨Cpolymerase chain reaction (RT-PCR) analysis of liver epithelial progenitor cell (LEPC) differentiation in culture. The expression of hepatocyte and bile duct markers was examined on LEPCs 2, 4, 8, and 10 after dimethylsulfoxide and Matrigel treatment (A). The PCR cycles were optimized to get the best contrast: ALB, 30 cycles; GS, 27 cycles; CK19, 26 cycles; Aquaporin-1, 30 cycles; GGT, 30 cycles; and HPRT, 26 cycles (as internal loading control). (B, C): Phase-contrast photographs of LEPC 2 on Matrigel supplement with 20 ng/ml hepatocyte growth factor (HGF). Two hours after plating (B) and three days after plating (C), LEPCs showed branching morphogenesis. Bar = 20 7mu;m.

Cultured LEPCs in three-dimensional Matrigel spontaneously formed aggregates, but the aggregates were transient and quickly disintegrated without EGF or HGF supplement. When exposed to the cytokine HGF (20 ng/ml), LEPCs in Matrigel gathered into small clones and formed tubule-like extensions, like so-called branching morphogenesis, within 3 days (Figs. 5B, 5C) . RNA analysis of the cell aggregates in Matrigel for 5 days revealed upregulation of biliary lineage markers CK19 and GGT, whereas the expression of CK8 and CK18 was unaltered (Fig. 5A). Another bile lineage molecule, aquaporin-1, undetectable in untreated cells, was also induced. Meanwhile, the expression of hepatocyte molecule GS was decreased relative to untreated cells (Fig. 5A).1 Y* @# o+ y* W) P! Y4 Y2 Z

When 50 ng/ml EGF was used instead of HGF, LEPCs again formed aggregates in Matrigel. However, unlike the HGF-dependent aggregates, the borders of EGF-dependent cell aggregates were smooth and without the tubule-like branches. RNA analysis of the cell aggregates 5 days after plating revealed robust upregulation of hepatocyte markers ALB and GS, down-regulation of CK19 relative to the untreated cells, and concomitant loss of GGT mRNA signal (Fig. 5A). To further follow the phenotype variation of LEPCs in Matrigel supplement with EGF, pools of CK19-EGFP and pAlb-DsRed-transfected LEPCs, in a 1:1 ratio, were seeded in Matrigel. Only green fluorescence was observed in the cell aggregates at first (Figs. 6A, 6B). Three days after seeding, the red fluorescence became detectable and progressively grew in strength and, concomitantly, the green fluorescence diminished (Figs. 6C, 6D). Ultra-structurally, these cells in the aggregates were homogeneous hepatocyte in appearance, with well-developed organelles such as mitochondria, Golgi apparatus, and parallel endoplasmic reticulum. Many bile canaliculi were observed in the intercellular space of adjacent cells (Figs. 6E, 6F).
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Figure 6. The phase-contrast photograph of the mixture of pCK19-EGFP and pAlb-DsRed-transfected liver epithelial progenitor cell (LEPC) 2 clones on Matrigel supplement with 50 ng/ml epithelial growth factor. The cells gathered into aggregates (A) and showed green fluorescence (B), but red fluorescence cannot be detected with same exposure time (data not shown). Five days after plating, the green fluorescence of the same cell aggregate diminished (C) and the red fluorescence was induced (D). Bile canaliculi were observed in the intercellular space of adjacent cells in aggregates by ultrastructural analysis (E). The cells in the aggregates were homogeneous hepatocytes with well-developed organelles (E, F). Arrowheads indicate tight junctions. Bar = 25 µm (A¨CD) and 2 µm (E, F).; c- o/ r4 G- D; P, _( i$ g( @' \4 W- j
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LEPC clonal lines 2, 4, 8, and 10 were all subjected to the above experiments, and responses were similar in all of these lines. The other six lines were not examined. Taken together, these results indicate that LEPCs are bipotential progenitors with the capacity to differentiate into bile duct or hepatocyte lineages when cultured in vitro in the presence of the appropriate inducing agents.5 z  M4 V# S4 L  y- I& [
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Engraftment of LEPCs into the Liver Parenchyma
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To assess the ability of LEPCs to engraft and to differentiate within the microenvironment of the liver in vivo, we prepared recipient animals by using the CCl4 protocol frequently used to elicit acute liver injury in recipients for cell transplantations . LEPC clonal line 2 transfected with pCK19-EGFP and pAlb-DsRed was used to transplant into CCl4-treated animals. The mice were killed 4 weeks (n = 4) and 6 months (n = 5) after implantation to detect transplanted cells (one mouse died after the initial surgery). No tumors were detected in any of the experimental animals. DsRed-positive cells, indicating engrafted LEPCs differentiated into hepatocyte lineage, were detected in the recipient mice at 4 weeks and 6 months after transplantation. The percentage of DsRed-positive cell engraftment was 2.1 ¡À 1.5 (n = 4) at 4 weeks and declined to 0.4 ¡À 0.5 (n = 5) by 6 months. At 4 weeks after transplantation, donor-derived cells were observed, usually embedded in liver parenchyma near the periportal area. At 6 months after transplantation, most of the donor-derived cells were single binucleated cells near the central vein (Figs. 7A, 7B), suggesting no obvious proliferation of LEPCs after transplantation. The overlay image of DsRed and serial section stained by anti--1 anti-trypsin antibody (green) clearly shows that all DsRed-positive donor cells were also -1 anti-trypsin-positive hepatocytes (yellow) 6 months after cells transplantation (Figs. 7C, 7D). Taken together, these observations demonstrate the ability of LEPCs to engraft and differentiate into hepatocyte lineage in vivo after transplantation into recipient animals.
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Figure 7. Engraftment and differentiation of liver epithelial progenitor cells (LEPCs) transplanted into CCl4-treated recipient mice. pAlb-DsRed-transfected LEPC 2 was transplanted into mice livers by spleen injection. DsRed-positive cells were detected in experimental mice 4 weeks and 6 months after cell transplantation with percent DsRed-positive cell engraftment of 2.1 ¡À 1.5 (n = 4) and 0.4 ¡À 0.5 (n = 5), respectively (A, B). Overlay image of DsRed and serial section stained by -1 anti-trypsin antibody (green) clearly shows that all EGFP-positive donor cells were also -1 anti-trypsin-positive hepatocytes (yellow) 6 months after cell transplantation. (D): DAPI staining of the same vision field of (C) to reveal nuclei. Relative magnification, x200.. }; m, Y) ~2 r) X8 e& a$ Q
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DISCUSSION
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In this report, we document that in adult mouse liver injured by Rs/PH, obvious oval cell¨Clike cell clusters arose near the portal tracts. We report the establishment of LEPC lines from Rs/PH-treated livers showing distinct ductular reactions. The bipotency of these liver progenitor cells was implicated by the expression of markers for both hepatocytes and biliary epithelial cells and, after seeding into Matrigel, differentiated into either bile lineage or hepatocyte lineage when exposed to HGF or EGF, respectively." n' n  H/ g6 `9 {1 e2 ^' L
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Fetal liver progenitors (e.g., hepatoblasts) are not known to express hematopoietic antigens such as CD45, CD34, and Thy-1. The isolated exception among hematopoietic markers is Sca-1, which is present in mouse-embryonic-day-12.5 liver epithelial cells . Our data show that LEPCs, which were isolated from adult livers showing ductular reactions, express c-Kit and Thy-1 but not CD34 and CD45. At present, the precise origin of the oval cell¨Clike LEPCs remains to be established. However, the expression of the hematopoietic markers by LEPCs may be of biologic significance and needs to be further investigated.; q1 E1 x) G8 l; p

To investigate lineage plasticity of the liver progenitors, we constructed expression vectors with the albumin promoter controlling DsRed (pAlb-DsRed) and CK19 promoter controlling EGFP (pCK19-EGFP) to monitor differentiation along the hepatocyte or the biliary lineages, respectively. This two-color differentiation reporter system is more versatile, easier to handle, and overcomes some of the common problems associated with immunohistochemical approaches to monitor protein markers . In contrast, when exposed to 50 ng/ml EGF, LEPCs differentiated along the hepatocyte lineage. In the absence of cytokine supplement, cell aggregates formed but disintegrated within 48 hours, whereas RNA analysis showed transient upregulation of hepatocyte-associated molecules (data not shown). Quite interestingly, our preliminary data further suggest that EGF may not be sufficient to elicit differentiation into hepatocytes because exposure to both EGF and HGF resulted only in tubule-like structures and differentiated only toward biliary lineage (data not shown). Taken together, these results suggest that EGF can maintain LEPC survival in Matrigel but not necessary to induce hepatocyte differentiation.6 y+ X3 b7 K8 g4 v8 v# w5 \
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The ability of LEPCs to differentiate into hepatocyte lineage was best demonstrated in vivo within the hepatic microenvironment. Recipient animals preconditioned by CCl4 and infused with pAlb-DsRed-transfected LEPCs showed DsRed-positive cells in the liver. On the other hand, no EGFP-positive bile lineage cells were found in the recipient liver after transfusion with pCK19-EGFP LEPCs. At present, there remains a very remote possibility that the DsRed signal in the liver is the result of cell fusion events after transplantation, but to our knowledge, there is no published evidence supporting fusion of liver progenitor cells with host hepatocytes.* S9 m% a& K5 R) Z2 O1 _2 U8 M9 {$ [4 }

The LEPCs, when maintained in the presence of other cells (e.g., before purification of the epithelial population by removing mesenchymal cells) (Figs. 1C, 1D), exhibited a typical morphology very similar to hepatic progenitors described by others. It is noteworthy, however, that upon progressive purification, the cloned LEPCs adopted an altered appearance that was less hepatic parenchymal-like while maintaining the expression of the hepatic parenchymal markers. It is possible that mesenchymal cells contributed to the overall characteristics of these liver progenitor populations, and this is currently under investigation.  t8 C$ x5 W9 P8 K+ ]

Another interesting aspect of the LEPCs was the nonuniform distribution of CK19, as revealed by anti-CK19 immunostaining (Fig. 2A). As an endogenous cytoskeleton component, CK19 was supposed to be located universally and evenly in cytoplasm. To our knowledge, the polarized distribution of CK19 in cultured liver progenitor cells or bile duct cells in situ has not been reported previously. However, CK19 molecule has been found in the apical but not cytoplasmic compartment of mouse (but not human) pancreas acinar cells . More detailed studies will be needed to define the biological significance of this observation./ b9 g8 l3 @8 g1 i6 Y) Z

With the availability of powerful genetics and extensive disease models, the use of the mouse to study liver regeneration after injury and liver progenitor cells is highly desirable. For example, use of oval cells derived from liver-associated gene-modified mice is a powerful approach to explore the mechanism of liver development and regeneration. However, for reasons that are not entirely clear, the isolation of murine hepatic stem cells is comparatively difficult among all of the mammalian species studied. Many reliable and reproducible methods to induce rat liver progenitor cells frequently do not work as well in the mouse . More specifically, in our opinion, the 2/3 PH can concisely remove the median and left lateral lobes of the mouse liver. However, tissue necrosis in the remaining liver mass due to the 2/3 PH can be quite variable among animals. Because of the relatively smaller original liver mass in the mouse compared with the rat, the inconsistent degree of tissue necrosis might have a significantly greater impact on the overall loss of liver mass, and this in turn might contribute to differing liver regenerative kinetics and induction of mouse oval cell progenitors.( w4 _  u3 I# ]& x' g

Finally, oval cells is a name that only reflects their morphology; their origin, heterogeneity, and biologic roles remain ambiguous. The LEPCs lines described here are apparently derived from the oval cell compartment. However, the combined and unusual properties of these LEPCs, particularly in the expression of c-Kit and Thy 1, render them distinct from previous oval cell lines. Hence, these LEPC lines hold promise as unique and novel models of cellular plasticity and hepatocarcinogenesis.
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ACKNOWLEDGMENTS  b5 y9 ]* ]2 f, M% J7 R

This work was supported by the National Nature Science Foundation (grants 30270668 and 30200138), Shanghai Key Basic Science Project (grant 03DJ14020), and The Program for Backbone College Teachers issued by the Education Ministry of China. We thank Shi Lvji (Chinese Academy of Science), Zeng Yitao (Shanghai Institute of Medical Genetics), and Sheng Huizen (Shanghai Second Medical University) for useful discussion. We also thank Zhang Jun, Ph.D. (Changhai Hospital, Second Military Medical University), for technical assistance in FACS analysis, Prof. Derek LeRoith for providing the plasmid pGEMAlbSVPA, and Prof. R. Kemler for TROMA-3 monoclonal anti-CK19 antibody.# f5 _' Y% \# c& r; S/ z& w
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DISCLOSURES* _5 D* t) B9 i# [/ V0 a  B, k9 r

The authors indicate no potential conflicts of interest.
      【参考文献】

Watt FM, Hogan BLM. Out of Eden: Stem cells and their niches. Science 2000;287:1427¨C1430.
/ N9 Z( _; d1 i* Y6 n. f0 T; Y
Weissman IL. Translating stem and progenitor cell biology to the clinic: Barriers and opportunities. Science 2000;287:1442¨C1446.6 W" Q' v! C: u- {7 @) v2 h# P9 |
9 t* c1 B3 T/ @5 F
Shiojiri N, Lemire JM, Fausto N. Cell lineages and oval cell progenitors in rat liver development. Cancer Res 1991;51:2611¨C2620.
8 Z6 t+ G7 I' `2 q0 `; q$ O
Rogler LE. Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro. Am J Pathol 1997;150:591¨C602.1 y# M9 }0 e8 z, M: t/ ]( F# r

Kubota H, Reid LM. Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci U S A 2000;97: 12132¨C12137.4 D% u; s- y- O+ Y! N

Suzuki A, Zheng YW, Kondo R et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 2000;32:1230¨C1239.
' D- ~7 s$ i; c
Suzuki A, Zheng YW, Kaneko S et al. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 2002;156:173¨C184." s) H- L6 r( ]+ F/ R4 _

Marchand HS, Weiss MC. Inducible differentiation and morphogenesis of bipotential liver cell lines from wild-type mouse embryos. Hepatology 2002;36:794¨C804.9 l; {' K3 H& M; [) M
4 C$ y+ E2 A0 q2 ^
Malhi H, Irani AN, Gagandeep S et al. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002;115:2679¨C2688.
$ ]" _( b1 Q7 L, @- r6 [
Germain L, Blouin MJ, Marceau N. Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components. Cancer Res 1988;48:4909¨C4918.$ }# y. t4 e; j

Thorgeirsson SS. Hepatic stem cells in liver regeneration. FASEB J 1996;10:1249¨C1256.

Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276:60¨C66.
. `+ d4 G  T+ E& f
Farber E. Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene. Cancer Res 1956;16:142¨C155.

Lemire JM, Shiojiri N, Fausto N. Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine. Am J Pathol 1991;139:535¨C552.) Q0 `( `  H: m5 v+ g) x; o6 m( x
. X: ^# P& `; g2 n: f
Germain LM, Gourdeau NH, Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res 1988;48:368¨C378.0 b5 e1 a7 e* C; U: s0 f( D( c

Evarts RP, Hu Z, Fujio K et al. Activation of hepatic stem cell compartment in the rat: Role of transforming growth factor , hepatocyte growth factor, and acidic fibroblast growth factor in early proliferation. Cell Growth Differ 1993;4:555¨C561.0 n! f( j0 z: [3 Q' _

Laconi E, Oren R, Mukhopadhyay DK et al. Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol 1998;153:319¨C329.4 F6 I4 a# ^0 i

Gordon GJ, Coleman WB, Hixson DC et al. Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol 2000;156:607¨C619.

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¨C786.
5 h* H0 z; z4 H( P. l, }. Z7 g  b5 c! f7 j
Sell S. Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 2001;33:738¨C750.( [9 H- ]/ e: l+ o. p
& m4 J6 \  Y1 q
Gordon GJ, Butz GM, Grisham JW et al. Isolation, short-term culture and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats. Transplantation 2002;73:1236¨C1243.

Dahlke MH, Popp FC, Bahlmann FH et al. Liver regeneration in a retrorsine/CCl4¨Cinduced acute liver failure model: Do bone marrow-derived cells contribute? J Hepatol 2003;39:365¨C373.

Richards WG, Yoder BK, Isfort RJ et al. Isolation and characterization of liver epithelial cell lines from wild-type and mutant TgN737Rpw mice. Am J Pathol 1997;150:1189¨C1197.  v( k0 B5 F8 N! O. R1 q

Isfort RJ, Cody DB, Stuard SB et al. The combination of epidermal growth factor and transforming growth factor-beta induces novel phenotypic changes in mouse liver stem cell lines. J Cell Sci 1997;110:3117¨C3129.

Wang X, Foster M, Al-Dhalimy M et al. The origin and liver repopulating capacity of murine oval cells. Proc Natl Acad Sci U S A 2003; 100(suppl 1):11881¨C11888.

Vemuru RP, Aragona E, Gupta S. Analysis of hepatocellular proliferation: Study of archival liver tissue is facilitated by endogenous genetic markers of DNA replication. Hepatology 1992;16:968¨C973.% c/ z4 K* [2 K) w( j5 _
' g3 ^  p  B, ^* E/ n
Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976;13:29¨C83.9 R% X9 V$ E  H, T$ Y9 H
- l4 B& V0 F3 K+ H" t
Li J, Ning G, Duncan SA. Mammalian hepatocyte differentiation requires the transcription factor HNF-4. Genes Dev 2000;14:464¨C474.

Brembeck FH, Rustgi AK. The tissue-dependent keratin 19 gene transcription is regulated by GKLF/KLF4 and Sp1. J Biol Chem 2000;275: 28230¨C28239.3 `. h) e, l* V. }3 ]7 E

Rajvanshi P, Kerr A, Bhargava KK et al. Studies of liver repopulation using the dipeptidyl peptidase IV deficient rat and other rodent recipients: Cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule. Hepatology 1996;23: 482¨C496.$ \6 ?9 L* X# w. Z
- O+ {/ M: |/ r! I' D# Y, g
Gupta S, Rajvanshi P, Lee CD. Integration of transplanted hepatocytes in host liver plates demonstrated with dipeptidyl peptidase IV deficient rats. Proc Natl Acad Sci U S A 1995;92:5860¨C5864.2 t3 y+ c+ M. Z& v& I& `

Baumann U, Crosby HA, Ramani P et al. Expression of the stem cell factor receptor c-Kit in normal and diseased pediatric liver: Identification of a human hepatic progenitor cell? Hepatology 1999;30:112¨C117.* r4 F1 ]+ Y1 y! g$ S% i

Lijun Y, Shiwu L, Heather H et al. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99:8078¨C8083.

Petersen BE, Goff JP, Greenberger JS et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 1998;27: 433¨C445.

Omori N, Omori M, Evarts RP et al. Partial cloning of rat CD34 cDNA and expression during stem cell-dependent liver regeneration in the adult rat. Hepatology 1997;26:720¨C727.
7 q" y4 O. O' [4 Z$ x8 v: q
Spagnoli FM, Amicone L, Tripodi M et al. Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cytomet in the liver. J Cell Biol 1998;143:1101¨C1112.- d! p/ f% E, M; K7 I9 s1 a5 i/ L2 M
; L3 N: c' o. w  {$ X: g% A
Li Y, Sun MZ, Zoran L et al. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats. Hepatology 2002;35:315¨C324.
% I' e) M7 I4 w5 O( J
Ros¨¢rio M, Birchmeier W. How to make tubes: Signaling by the Met receptor tyrosine kinase. Trends Cell Biol 2003;13:328¨C335.

Yamamoto H, Quinn G, Asari A et al. Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application. Hepatology 2003;37:983¨C993." t) Z% R; B( X

Nierhoff D, Ogawa A, Oertel M et al. Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology 2005;42:130¨C139.
8 T0 u- A; ^: _) @8 {9 o# p
Petersen BE, Grossbard B, Hatch H et al. Mouse A6¨Cpositive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology 2003;37:632¨C640.$ a* ^) N% c7 X- U4 }
9 U) ?. |, F7 |8 L" w$ w/ k0 @, j
Matsusaka S, Tsujimura T, Toyosaka A et al. Role of c-Kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminoflu-orene/partial hepatectomy model. Hepatology 1999;29:670¨C676.& [* r6 I2 _, M1 M* U" m7 |( z
% ?0 e* }8 ?7 `6 }
Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168¨C1170.
7 U+ ?3 g% [% E8 j  |
Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897¨C901.% F9 S! M% K/ X, E+ m
. ~& e/ }( d5 ]- H
Willenbring H, Bailey AS, Foster M et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004;10:744¨C748.
, Q. E# _! Y3 W+ d/ x" h
Grompe M. The role of bone marrow stem cells in liver regeneration. Semin Liver Dis 2003;23:363¨C372.

Rajagopal J, Anderson WJ, Kume S et al. Insulin staining of ES cell progeny from insulin uptake. Science 2003;299:363.- h4 p) _% a/ E; y* ^

Grompe M. The importance of knowing your identity: Sources of confusion in stem cell biology. Hepatology 2004;39:35¨C37.
6 f! {0 D8 x9 M
Kawaida K, Matsumoto K, Shimazu H et al. Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proc Natl Acad Sci U S A 1994;91:4357¨C4361., m5 x( {' X$ l9 B& ?- p- o
8 j2 K5 Y; I. ?' r4 j% J4 [' T8 K
Ku NO, Zhou X, Toivola DM et al. The cytoskeleton of digestive epithelia in health and disease. Am J Physiol 1999;277:G1108¨CG1137.& p5 z; p; @% V

Lee JH, Ilic Z, Sell S. Cell kinetics of repair after allyl alcohol-induced liver necrosis in mice. Int J Exp Pathol 1996;77:63¨C72.; Q0 {9 s8 |  p4 b3 a

Guo DQ, Fu T, Nelson JA et al. Liver repopulation after cell transplantation in mice treated with retrorsine and carbon tetrachloride. Transplantation 2002;73:1818¨C1824.7 f2 g6 n: C2 E7 ^( M

Akhurst B, Croager EJ, Farley-Roche CA et al. A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver. Hepatology 2001;34:519¨C522.

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