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TISSUE-SPECIFIC STEM CELLS

Long-Term Culture of Postnatal Mouse Hepatic Stem/Progenitor Cells and Their Relative Developmental Hierarchy

^aDepartment of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;
^bDivision of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan;
^cDepartment of Gastroenterology, Juntendo University School of Medicine, Izunokuni, Japan

Key Words. Side population cells • Sca-1+ cells • Serum-free medium • Long-term culture Fluorescence-activated cell sorting analysis • Hepatic stem cells

Correspondence: Tatsutoshi Nakahata, M.D., Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: +81-75-751-3290; Fax: +81-75-752-2361; e-mail: tnakaha{at}kuhp.kyoto-u.ac.jp

Received September 3, 2006; accepted for publication December 27, 2006.
First published online in STEM CELLS EXPRESS   January 11, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature -fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1+ cells. As mice aged, the frequency of SP and sca-1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%–40% of the SP cells expressed sca-1, but only a few sca-1+ cells were also SP cells. Analysis of colonies derived from single SP or sca-1+ cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1+ cells, whereas sca-1+ cells generated only sca-1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca-1+ cells. In regenerating livers, ABCG2+ cells and sca-1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Serial cell transplantation studies [1, 2] have revealed that postnatal livers contain populations that can expand over long periods. It remains unclear, however, where in the liver these hepatic stem cells reside. Moreover, although there is evidence for the existence of extrahepatic stem cells, such as bone marrow cells [3] and peripheral blood cells [4], that can travel to the liver and act as hepatic stem cells, it is unclear whether these cells transdifferentiate or fuse with hepatocytes to exert hepatic stem cell function. In addition, although postnatal hepatic stem/progenitor cells have been characterized by several markers, including CD34 [5, 6], c-kit [7–9], Thy-1 [10], sca-1 [11, 12], and side population (SP) cell attributes [12, 13], the location of each marker in the developmental hierarchy of hepatic stem/progenitor cells is not known. Answers to these questions are needed to devise novel strategies for cell transplantation or effectively induce endogenous hepatic stem/progenitor cells required for rapid recovery from severe liver damage. However, progress is hampered by difficulties in maintaining and expanding postnatal hepatic stem/progenitor cells and, consequently, few studies report long-term culture of postnatal liver cells. For example, in mouse, most reports analyze cells from transgenic or mutant mouse donors, namely Met murine hepatocyte cells [14, 15], which are generated from liver of mice expressing an active truncated form of the human Met receptor or from p53 null-transformed hepatic progenitor cells [16]. Two additional studies have sought to establish long-term cultures of hepatic cells from normal postnatal livers. In one, Azuma et al. [17] were relatively successful in maintaining mouse adult hepatic stem/progenitor cells by initially suspending hepatic cells in hypoxic conditions and then allowing them to form aggregates. In the other study, Block et al. have developed hepatocyte growth medium [18], a serum-free medium allowing normal adult hepatocyte expansion. However, neither of these methods permits extensive expansion of adult mouse hepatic stem/progenitor cells with repeated passage. With regard to the long-term culture of postnatal human liver cells, Parent et al. established HepaRG cell lines [19] with dual differentiation potential from a liver associated with chronic hepatitis C infection. However, cell lines with dual differentiation potential have not been established from normal postnatal human livers. Finally, long-term culture of postnatal liver cells from rats has been successfully achieved, including WB-344 [20], rat liver epithelial cells [21], and hepatic stem-like epithelial cells [22, 23]. However, markers of all of these cells have not been comprehensively assessed, and thus their place in the stem cell developmental hierarchy remains unknown.

Recently, we established a long-term culture system that permits extensive expansion of normal mouse fetal hepatic stem/progenitor cells [12]. This system employs serum-free medium containing B27, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). This system generates large colonies more readily than previously reported systems. The method utilizes a replating procedure employing diluted trypsin with knockout serum replacement and CaCl₂, thus maintaining cell-cell contact and viability. The resulting expanded fetal hepatic stem/progenitor cell populations are a heterogeneous mixture of cells including SP (5%–20%) and sca-1+ (approximately 15%) cells. Using these two stem cell markers, we could further enrich the population of fetal hepatic stem/progenitor cells.

In this study, we modified the culture system described above and succeeded in expanding postnatal hepatic stem/progenitor cells from undamaged postnatal livers. These cells could be maintained over the long periods by passage and are a heterogeneous mixture of cells that include SP and sca-1+ cells. Our previous study suggested that these cells have stem cell characteristics [12], and, indeed, we show here that both types proliferate and have bipotential differentiation ability. We also analyzed the place in the hierarchy of stem cell development that these cells occupy by purifying single SP and sca-1+ cells and evaluating their progeny. Finally, we examined the in vivo expression of the SP phenotype or sca-1 in normal and damaged livers.

	MATERIALS AND METHODS

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Mice
C57BL/6 mice (4-, 8-, 12-week-old; n = 5 per age group) were obtained from SLC (Hamamatsu, Japan, http://www.jslc.co.jp). Liver tissue served as the source of postnatal hepatic stem/progenitor cells. Regenerating livers were induced by injecting 4-week-old C57BL/6 mice intraperitoneally three times at 2-day intervals with anti-mouse Fas (0.3 mg/kg) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) [24] dissolved in phosphate-buffered saline (PBS). Five days after the last injection, regenerating livers were removed and analyzed. Mice were maintained according to Animal Protection Guidelines of Kyoto University (Kyoto, Japan).

Hepatic Cell Culture
Hepatic cells were dissociated using a 2-step collagenase method, collected by centrifugation at 500 rpm for 2 minutes, suspended in Dulbecco's modified Eagle's medium/F12 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) with B27 (Gibco, Grand Island, NY, http://www.invitrogen.com), ITS-X (Gibco), 10 mM HEPES (Nacalai Tesque Inc., Kyoto, Japan, http://www.nacalai.co.jp/en), antibiotics, 20 ng/ml EGF (Sigma-Aldrich), 20 ng/ml bFGF (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and 10 ng/ml HGF (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and finally plated onto 6-cm-diameter type 1 collagen-coated dishes (1 x 10⁶ cells per dish). This standard medium was changed every 3 days. On day 14, cells were harvested using diluted trypsin [12] and plated onto newly prepared collagen-coated dishes. On day 21, the colonies that contained more than 200 cells were counted and expanded.

Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was prepared by using TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and reverse-transcribed using a SuperScript first-strand synthesis system (Invitrogen). cDNA was amplified by polymerase chain reaction (PCR) with the AmpliTaq Gold kit (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com), and PCR was performed with primers specific for transcripts encoding the following proteins: the immature cell marker -fetoprotein (AFP); hepatocyte-specific markers albumin (ALB), 1-antitrypsin (1-A), glucose-6 phosphatase (G-6-P) [25], tyrosine amino transferase (TAT) [26], and tryptophan-2,3-dioxygenase (TO); cholangiocyte-specific markers cytokeratin (CK)19 and biliary glycoprotein (BGP) [25]; and internal control ß-actin (ß-act). The oligonucleotide primers were: AFP: 5'-ACT CAC CCC AAC CTT CCT GTC-3' 5'-CAG CAG TGG CTG ATA CCA GAG-3', ALB: 5'-CAT GAC ACC ATG CCT GCT GAT-3' 5'-CTC TGA TCT TCA GGA AGT GTA-3', 1-A: 5'-TCG ATC CTA AGC ACA CTG AGG-3' 5'-CGG CTT GTA AGA CTG TAG C-3', G-6-P: 5'-AAC CCA TTG TGA GGC CAG AGG-3' 5'-TAC TCA TTA CAC TAG TTG GTC-3', TAT: 5'-TCC AGG AGT TCT GTG AAC AGC-3' 5'-AGT ATA TGG TGC CTG CCT GC-3', TO: 5'-TGC GCA AGA ACT TCA GAG TGA-3' 5'-AGC AAC AGC TCA TTG TAG TCT-3', CK19: 5'-GTC CTA CAG ATT GAC AAT GC-3' 5'-CAC GCT CTG GAT CTG TGA CAG-3', BGP: 5'-GAA CTA GAC TCT GTC ACC CTG-3' 5'-GCC AGA CTT CCT GGA ATA GA-3', and ß-act: 5'-ATC CTG ACC CTG AAG TAC CCC ATT-3' 5'-CCA AGA AGG AAG GCT GGA AAA GAG-3'. The following conditions were employed for amplification: initial denaturation at 95°C for 9 minutes followed by 28 (ALB, AFP, and CK19), 30 (ß-act), or 35 cycles (others) of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and a final extension step at 72°C for 10 minutes. PCR products were then separated on 1.0% agarose gels.

Flow Cytometry
For fluorescence-activated cell sorting (FACS), we used the following markers: rat and human oval cell markers CD34 [5, 6], Thy-1 [10], and c-kit [7–9]; the 6 integrin subunit CD49f [25]; the hematopoietic marker CD45 [27]; the vascular endothelium marker CD31 [28]; the early mesodermal marker flk-1 [29]; and the stem/progenitor cell marker sca-1 [11, 12]. We also used FACS to identify SP cells [13], identifiable by their ability to exclude dyes such as Hoechst due to the expression of the transporter proteins ABCG2/BCRP1 [30]. For FACS analysis, cells (6–12 weeks after plating) from 4-week-old mice were harvested with cell recovery solution (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) for 20 minutes. The resulting cells were incubated with anti-mouse CD16/32 antibodies (BD Pharmingen) for 15 minutes on ice to block nonspecific binding and then incubated with fluorescein isothiocyanate (FITC)-conjugated sca-1, CD34, and Thy-1 antibodies, phycoerythrin (PE)-conjugated CD49f, CD31, and flk-1 antibodies, or allophycocyanin (APC)-conjugated c-kit and CD45 antibodies (BD Pharmingen) for 30 minutes on ice. FITC-conjugated rat IgG2a antibodies, PE-conjugated rat IgG2a antibodies, or APC-conjugated rat IgG2b antibodies (BD Pharmingen) were served as isotype controls. Dead cells were excluded by propidium iodide gating. Hoechst staining was performed as described previously [12]. After washing, the cells were analyzed or sorted by using FACSCalibur, BD LSR, or FACSVantage with the CellQuest program (Becton, Dickinson and Company). To analyze the relative developmental hierarchy of putative stem cells, expanded cells derived from 4-week-old mice were fractionated into four subpopulations using FACSVantage: SP sca-1– cells (group 1), SP sca-1+ cells (group 2), main population (MP) sca-1+ cells (group 3), and MP sca-1– cells (group 4). Our usual sorting procedure yielded purities of approximately 90%–95%. Single cells of each fraction were cultured in individual wells of 96-well type 1 collagen-coated dishes and confirmed microscopically. On day 14 after sorting, large colonies occupying more than 30% of the well surface were counted. Finally, five colonies representing SP sca-1– cells (group 1) and MP sca-1+ cells (group 3) were randomly collected and expanded. On day 24 after sorting, the frequency of SP cells and sca-1+ cells was determined and reverse transcription (RT)-PCR was performed with AFP, ALB, and CK19 primers.

Differentiation Culture Condition
To induce the differentiation of hepatocytic lineage cells, cells from 4-week-old mice that had been expanded for more than 6 weeks were employed. Expanded cells (5 x 10⁵ cells per milliliter) were cultured with standard medium plus 10 ng/ml oncostatin M (OSM; R&D Systems) [31, 32]. OSM at that concentration was added every 3 days when the medium was changed. On day 6, RT-PCR, periodic acid-Schiff (PAS) staining, and immunocytochemistry for carbamoyl phosphate synthetase I (CPSI; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) [33] were performed. On day 9, the frequencies of SP cells, and sca-1+ cells were determined using FACSCalibur, BD LSR, and FACSVantage.

Immunochemical Staining
Immunocytochemistry was performed as described previously [12]. The primary antibodies used were: goat anti-mouse ALB (Bethyl Inc., Montgomery, TX, http://www.bethyl.com), rabbit anti-human AFP (ICN Biochemicals, Costa Mesa, CA, http://www.mpbio.com), mouse anti-CK7 (Progen Industries Limited, Queensland, Australia, http://www.progen.com.au), goat anti-CPSI, and rat anti-mouse sca-1 (BD Pharmingen). Secondary antibodies used were FITC-conjugated donkey anti-goat IgG, Cy3-conjugated donkey anti-goat IgG, FITC-conjugated donkey anti-rabbit IgG, Cy3-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-mouse IgG, and Cy3-conjugated donkey anti-rat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Normal IgG of the species from which primary antibodies had been obtained served as primary antibodies in the negative controls. PAS staining was performed as described [34]. For immunohistochemistry, livers were collected, fixed in 10% formalin, and embedded in paraffin blocks. Sections 4-µm thick were cut and placed on silane-coated slides. After removing paraffin, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english) for 10 minutes at room temperature, and sections were incubated with primary antibodies diluted with PBS containing 0.1% saponin. Primary antibodies used were rat anti-mouse sca-1, rabbit anti-ABCG2 (Kamiya Biomedical Company, Seattle, WA, http://www.kamiyabiomedical.com), mouse anti-CK19 (DAKO, Glostrup, Denmark, http://www.dako.com), and rabbit anti-cow CK (Z0622; DAKO), which detects bile ducts and oval cells [35]. Slides were then stained by using Histofine Simple Stain Max-PO (Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html) and DAB Substrate Kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) for sca-1, Vectastain ABC Kit and DAB Substrate Kit (Vector Laboratories) for anti-ABCG2 and anti-cow CK, and DAB Substrate Kit, M.O.M Kit, and Vectastain ABC Kit (Vector Laboratories) for anti-CK19. Nuclei were stained using Mayer's hematoxylin solution (Wako Chemical).

Statistical Analysis
The data are presented as mean ± SD. Student's t test was used to determine the statistical significance of observed differences.

	RESULTS

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Effective Expansion of Postnatal Hepatic Stem/Progenitor Cells and Analysis of Their Characteristics
We previously showed that we could extensively expand fetal hepatic stem/progenitor cells using our standard medium [12]. Therefore, we sought to expand postnatal hepatic stem/progenitor cells using the same medium. Hepatic cells originated from 4-, 8-, and 12-week-old male mice (n = 5, in each group). Initially, we directly cultured sorted cells obtained after liver dissociation; however, this procedure led to poor cell viability. Subsequently, we centrifuged a whole cell preparation at 500 rpm for 2 minutes, since our early studies showed that single fetal liver stem/progenitor cells that were expanded with our culture system could be collected by this centrifugation step. The resulting pellet consisted largely of mature hepatocytes, and the supernatant contained nonparenchymal cells. When the supernatant was cultured, the nonparenchymal cells, such as endothelial cells, hematopoietic cells, and stellate cells, showed rapid growth, but extensive expansion of hepatic stem/progenitor cells was not observed (data not shown). By contrast, when 1 x 10⁶ pellet cells were plated on 6-cm type 1 collagen-coated dishes, rapid growth of endothelial cells, hematopoietic cells, and stellate cells was not detected, but rather expansion of hepatic stem/progenitor cells was observed. The plated hepatic cells first attached to dishes as a monolayer, and some formed floating and attaching spheroids that did not grow. By day 14, after repeated medium changes at 3-day intervals, most cells forming monolayer sheets or spheroids had died, leaving small polygonal living cells (Fig. 1A). On day 14, the cells were passaged for the first time using diluted trypsin, which maintains cell-cell contact. Harvested cells were replated on collagen-coated dishes using our standard medium. On day 21, we determined the frequency of the dishes containing large colonies (more than 200 cells) and found that the frequency of the dishes obtained from the 4-, 8-, and 12-week-old mice was 40%, 20%, and 0%, respectively. During subsequent culturing, these large colonies gradually expanded (Fig. 1B, 1C), and by day 45, stable expansion of cells was observed (Fig. 1D). These stable expanding cells formed heterogeneous populations containing immature AFP+ cells, hepatocytic lineage cells (ALB+ cells), and cholangiocytic lineage cells (CK7+ cells) (Fig. 1E, 1F). The expanded cultures maintained these phenotypes after further passages. AFP+ cells were relatively smaller than the ALB+ cells and the CK7+ cells (Fig. 1E, 1F). Notably, whereas cells from 4-week-old mice could be maintained for over 30 passages, cells from 8-week-old mice could only be maintained for approximately 20 passages and then gradually lost their ability to expand. Flow cytometric analysis of expanded heterogeneous day 45 cells revealed that they expressed CD49f (+low) but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1 (Fig. 2A). This was true for both 4- and 8-week-old mouse-derived cultures. SP cells and/or sca-1+ cells were also found in heterogeneous expanded cell populations; however, the frequencies of SP cells and sca-1+ cells in the population varied depending on the age of the donor mouse. Thus, whereas 2%–4% and 15%–40% of the expanded cells from 4-week-old mice were SP and sca-1+, respectively, cell populations from 8-week-old mice showed fewer sca-1+ cells (approximately 1%) and no SP cells (Fig. 2B, 2C). Thus, our culture system can successfully generate long-term postnatal hepatic cell cultures containing cells expressing immature markers.

View larger version (73K): [in this window] [in a new window]   Figure 1. Culture and immunocytochemistry of expanded cells. (A–D): Time course of cell culture. These images were obtained from cultured cells from 4-week-old mice. (A): Cultures on day 14 just prior to the first passage: some small expanding cells can be seen. (B, C): Cultures on day 30: large colonies have formed. (D): Cultures on day 45: stably expanding cells can be detected. (E, F): Analysis of expanding cells shown in (D) with regard to -fetoprotein (AFP) expression ([E, F]; red), albumin (ALB) expression ([E]; green), AFP and ALB expression ([E]; yellow), and cytokeratin 7 expression ([F]; green) (original magnification: [B], x40; [A, C, E, F], x100; [D], x200).

View larger version (43K): [in this window] [in a new window]   Figure 2. Flow cytometric analysis of expanded cells. (A): Flow cytometric analysis of day 45 expanded cells showing that these heterogeneous cells are CD45–, CD34–, Thy-1–, c-kit–, CD31–, flk-1–, and CD49f+low+. Sca-1+ cells are also detected. (B, C): Day 45 expanded cells originating from 4-week-old mice include side population cells (B), unlike cultures derived from 8-week-old mice (C). Abbreviations: 4W, 4-week-old mice; 8W, 8-week-old mice; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

View larger version (50K): [in this window] [in a new window]   Figure 3. Induction of differentiation by oncostatin M (OSM) treatment. (A): Before adding OSM, expanded cells continued to express genes encoding AFP, ALB, 1-A, G-6-P, CK19, and BGP (lane 1). However, on day 6 after addition of OSM, hepatocytic differentiation markers TAT and TO are induced (lane 2). (B–E): Compared with untreated cells (B, D), OSM treatment generates cells strongly positive for periodic acid-Schiff (C) and carbamoyl phosphate synthetase I ([E]; red) by day 6. (F, G): Flow cytometric analysis indicates that, 9 days after adding OSM, the frequency of side population cells decreases (F, G), whereas that of sca-1+ cells increases ([H]; black line, isotype control; blue line, sca-1 positivity before adding OSM; red line, sca-1 positivity after adding OSM) (original magnification: [B–E], x200). Abbreviations: 1-A, 1-antitrypsin; ß-act, ß-actin; AFP, -fetoprotein; ALB, albumin; BGP, biliary glycoprotein; CK, cytokeratin; G-6-P, glucose-6 phosphatase; TAT, tyrosine amino transferase; TO, tryptophan-2,3-dioxygenase.

View larger version (30K): [in this window] [in a new window]   Figure 4. Flow cytometry, immunocytochemistry, and RT-PCR of SP and sca-1+ cells in the expanded populations derived from 4-week-old mice. (A–F): SP cells (A, B) are smaller than sca-1+ cells (D, E), and 20%–40% of SP cells are sca-1+ (A, C), whereas less than 5% of sca-1+ cells exhibit the SP phenotype (D, F). Immunocytochemistry shows that, whereas sca-1+ cells ([G–I]; red) rarely express AFP ([G]; green) or ALB ([H]; green), more than 20% of sca-1+ cells express CK7 ([I]; green). (J): The expanded cell population was fractionated into indicated subpopulations, which were then cultured at the single-cell level. Numbers of large colonies arising from fractionated cells by day 14 after sorting and plating were determined. This revealed that SP cells have an enhanced ability to form colonies, unlike sca-1+ cells. (K): Single cell culture of SP and sca-1+ cells. We randomly collected 1–5 large colonies from single SP sca-1– cells and 6–10 from single MP sca-1+ cells. Flow cytometric analysis revealed that, whereas the SP sca-1– cells give rise to both SP cells and sca-1+ cells, MP sca-1+ cells produce mainly sca-1+ cells. RT-PCR also reveals that, whereas both SP sca-1– and MP sca-1+ cells have bipotential differentiation potential, cells derived from SP sca-1– cells express higher levels of AFP than those derived from MP sca-1+ cells (original magnification: [G–I], x100). Abbreviations: AFP, -fetoprotein; ALB, albumin; CK, cytokeratin; FSC, forward scatter; MP, main population; PI, propidium iodide; RT-PCR, reverse transcription-polymerase chain reaction; SP, side population; SSC, side scatter.

SP Cells and Sca-1+ Cells Reside in the Periportal Area of Regenerating Livers
We next determined whether cells expressing SP and sca-1 stem cell markers are found in normal or regenerating livers by searching for ABCG2-expressing and sca-1-expressing cells. SP phenotype (the ability to exclude dyes) arises from the expression of the ATP-binding cassette transporter ABCG2/BCRP1 [36], and thus SP cells in the liver were identified on the basis of their ABCG2 expression. To induce immature hepatic cells, 4-week-old mice were injected three times at 2-day intervals with anti-mouse Fas (0.3 mg/kg) dissolved in PBS. Five days after the last injection, regenerating livers were removed and analyzed. Many ductular proliferating cells that were positive for anti-cow CK and exhibiting oval cell morphology were apparent (Fig. 5A–5D). The luminal surface of the ductular proliferating cells in the portal area strongly expressed ABCG2 (Fig. 5F). By contrast, in normal liver, only the canalicular membrane of hepatocytes weakly expressed ABCG2 (Fig. 5E). With regard to sca-1, sca-1 positivity in liver was not restricted to hepatic stem/progenitor cells but was also seen in endothelial cells, hematopoietic cells, and other cells including hepatic stem/progenitor cells (data not shown). Endothelial cells around the portal area in the regenerating liver strongly expressed sca-1 (Fig. 5H), whereas the same cells in the normal liver showed much weaker expression (Fig. 5G). In the regenerating liver, although sca-1-expressing cells were mainly endothelial cells, some terminal bile duct cells and interlobular bile duct cells also expressed sca-1 (Fig. 5H). Ductular proliferating cells expressing ABCG2 rarely expressed sca-1 (data not shown). These observations indicate that the portal area is a likely hepatic stem cells niche.

View larger version (168K): [in this window] [in a new window]   Figure 5. Immunohistochemical analysis of cytokeratin (CK)19, ABCG2, and sca-1 expression in normal and regenerating livers. (A, B): Histological analysis of normal liver (A) and regenerating liver damaged by anti-mouse Fas (B). Many ductular proliferating cells resembling oval cells are detected in damaged but not normal livers (B). (C, D): Immunohistochemical analysis of damaged liver indicates CK19+ bile duct cells (C) and ductular proliferating cells positive for anti-cow CK, which detects bile ducts and oval cells ([D]; arrows). (E, F): Immunohistochemical analysis of ABCG2 expression in normal (E) and damaged (F) livers. Hepatocytes in normal liver express ABCG2 (E), whereas the luminal surface of the ductular proliferating cells in regenerating liver expresses ABCG2 strongly ([F]; arrowheads). (G, H): Immunohistochemical analysis of sca-1 expression in normal (G) and regenerating (H) liver. In normal liver, sca-1+ endothelial cells surrounding the portal area are detected, whereas in regenerating livers, this expression was stronger, and some bile duct cells also express sca-1 (original magnification: [A–D, G, H], x200; [E, F], x400).

	DISCUSSION

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Hepatic Stem Cells with Highly Regenerative Potential Decrease with Age
In comparing the number of SP and sca-1+ cells, we found that there were fewer SP cells in the expanded postnatal hepatic stem/progenitor cell population (2%–4%) than in expanded fetal hepatic stem/progenitor cell populations (more than 5%). This observation suggests that the number of stem cells decreases with age. This idea is supported by observations that (a) SP cells could be expanded from livers of 4-week-old but not 8- or 12-week-old mice, (b) the expanded population from the livers of 8-week-old mice contained fewer sca-1+ cells (approximately 1%) than the 4-week-old liver derived population (15%–40%), and (c) although cells originating from 4-week-old mice could be maintained for over 30 passages, cells from 8-week-old mice could only be maintained for about 20 passages and gradually lost their ability to expand. Thus, we estimate that the number of stem cells with the greatest regenerative ability decreases with age as does liver regenerative potential.

The Periportal Area Is the Niche of Hepatic Stem/Progenitor Cells
We next analyzed which cells in the normal or regenerative livers of 4-week-old mice express the SP cell marker ABCG2 and which express sca-1. In the normal liver, some mature hepatocytes express ABCG2, although the main expression of sca-1 was by the endothelial cells around the portal area. In contrast, in regenerating livers, ductular proliferating cells expressed ABCG2 and some bile duct cells expressed sca-1. These results reveal that ABCG2+ and sca-1+ cells exist in the portal area (the putative hepatic stem cell niche). However, the distribution of ABCG2+ and sca-1+ cells differed. In particular, ABCG2+ cells were found in the vicinity of the canal of Hering, supporting a previous report suggesting that hepatic stem cells are located near this location. Notably, our immunocytochemical analysis revealed that sca-1+ cells also tended to express CK7+ more often than AFP and ALB, suggesting that sca-1+ cells are more likely to be of the cholangiocytic than the hepatocytic lineage.

Utility of Our Expanded Cell System
Our expanded cell populations may be useful in analysis of self-renewal and differentiation of hepatic stem cells. Recent studies showed that, although embryonic stem cells [41, 42] are attractive hepatic stem cell sources for such study, this cell source is problematic because it is difficult to differentiate highly enriched populations of hepatic cells. By contrast, our culture system expands only hepatic cells, making it ideal for characterizing various hepatic stem/progenitor cells in the liver and for assessing their relative position in a developmental hierarchy.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported by a Grant-in-Aid for Creative Scientific Research (13GS0009) and a Grant-in-Aid for Scientific Research (B) (15039219) from the Ministry of Education, Science, Technology, Sports and Culture of Japan.

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