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Salivary gland progenitor cells induced by duct ligation differentiate into hepatic and pancreatic lineages
LIVER BIOLOGY AND PATHOBIOLOGY
Salivary Gland Progenitor Cells Induced by Duct Ligation Differentiate Into Hepatic and Pancreatic Lineages Kenji Okumura,1 Kimitoshi Nakamura,1 Yuichiro Hisatomi,1 Koji Nagano,1 Yasuhiko Tanaka,1 Kunihiko Terada,2 Toshihiro Sugiyama,2 Kazuhiro Umeyama,3 Kozo Matsumoto,4 Tetsuro Yamamoto,5 and Fumio Endo1 Tissue damage can be assessed based on regenerative responses, including progenitor cell proliferation. In the salivary gland, tissue damage induced by ligation of main ducts leads to the disappearance of acinar cells and to marked proliferation of ductal cells. Reopening of the ducts leads to repopulation of acinar cells within 1 to 2 weeks, which suggests activation of tissue progenitor cells in a damaged state. Because submandibular glands derive from the endoderm and ectoderm, we investigated the possibility of the presence of endodermal progenitor cells. We cultured cells obtained from the ligated salivary gland and identified colonies of epithelium-like cells. We singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The SGP-1 expresses both ␣61 integrin and cytoplasmic laminin. The hematopoietic stem cell marker CD34 and hepatic oval cell markers such as albumin, ␣-fetoprotein (AFP), and cytokeratin 19 are all negative. However, when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into hepatic trabecula and produced albumin. When SGP-1 cells formed clusters on type I collagen-coated dishes, they differentiated into endodermal lineage and 2 major types of clusters appeared: one contained cells positive for AFP and/or albumin (hepatic cluster) and the other positive for glucagon and/or insulin (pancreatic cluster). On laminin-coated dishes, SGP-1 selectively differentiated into hepatic-type cells. In conclusion, the multipotent progenitor cells isolated from the rat salivary gland have characteristics of tissue stem cells and can differentiate into cells of endodermal lineages. (HEPATOLOGY 2003;38:104-113.)
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he oval cell, a tissue stem cell for liver cell types, can differentiate into hepatocytes and bile duct epithelium when transplanted into the liver.1,2 These cells are a heterogeneous population positive for both hepatocyte and bile ductal cell markers such as albuAbbreviations: AFP, ␣-fetoprotein; 2-AAF, 2-acetylaminofluorene; LEA, LongEvans agouti; PCR, polymerase chain reaction; EGFP, enhanced green fluorescent protein; PAS, periodic acid–Schiff; H-CFU-C, hepatic colony-forming units in culture. From the 1Department of Pediatrics, School of Medicine, and the 5Division of Molecular Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2Department of Biochemistry, Akita University School of Medicine, Akita, Japan; 3Department of Cell Biology, Bios Inc., Kamakura, Japan; and 4Institute for Animal Experimentation, University of Tokushima School of Medicine, Tokushima, Japan. Received February 13, 2003; accepted March 29, 2003. Supported in part by grants for Research for the Future Program of JSPS, grantin-aid for Scientific Research and grant-in-aid for 21st Century COE Research from the Ministry of Education, Science, Technology, Sports and Culture, “Cell Fate Regulation Research and Education Unit,” and a research grant from the Ministry of Health, Labour and Welfare. Address reprint requests to: Fumio Endo, M.D., Ph.D., Department of Pediatrics, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 8608556, Japan. E-mail: [email protected]; fax: (81) 96-366-3471. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3801-0015$30.00/0 doi:10.1053/jhep.2003.50259 104
min, ␣-fetoprotein (AFP), cytokeratin 19, and other bile duct antigens.3 Oval cell populations also contain hematopoietic cell surface antigen–positive cells. The Thy-1 highly positive oval cell can differentiate into pancreatic cell types that secrete insulin, glucagon, and pancreatic polypeptide when cultured in the presence of high concentrations (23 mmol/L) of glucose.4 In the liver, after two-thirds hepatectomy, the regeneration process rapidly restores the original mass of the liver.5 During this process, oval cells do not appear and regeneration of the liver after hepatectomy is not likely to depend on tissue stem cells. In contrast, oval cells are seen during the process of progenitor-dependent liver regeneration.6 Progenitor-dependent liver regeneration has been shown under several experimental conditions. Administration of 2-acetylaminofluorene (2-AAF) combined with partial hepatectomy is one representative protocol.7 Under these conditions, 2-AAF suppresses hepatocyte proliferation and oval cells appear in the periportal area before restoration. These observations on hepatic oval cells imply that multipotent progenitor cells are induced during tissue injury in various organs. The pancreas is an organ of endodermal origin, and tissue injury can lead to the appearance of multipotent
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stem cells for the liver and the pancreas. When weaning rats are fed a copper-deficient diet for 8 weeks, these manipulations result in a marked reduction in the acinar mass and cytokeratin 19 –positive duct-like epithelial cells proliferate.8 If they are refed copper, cells with multiple hepatocellular characteristics emerge from the remaining pancreatic ducts.9 This work has been interpreted to suggest the presence of a pancreatic liver stem cell,10 and Dabeva et al. reported that pancreatic epithelial progenitor cells isolated from the copper-deficient diet rat pancreas can differentiate into hepatocytes when transplanted into the liver.8 Recent studies have shown that there is a tissue stem cell in the pancreas that can differentiate into hepatocytes in addition to pancreatic cell types. Zulewski et al. reported that multipotent stem cells from rat and human islets differentiated into hepatic cell types in culture when the cell density increased.11 In other experiments, a cell line from pancreatic acinar cells differentiated into hepatocytes in the presence of dexamethasone.12,13 In addition, cells including the pancreatic duct area repopulated in the liver.14 Thus, tissue stem cells isolated from liver and pancreas can undergo trans-differentiation to cells of other tissues.15,16 The salivary gland is derived from the endoderm, and the ectoderm participates in organogenesis.17 Salivary glands consist of many cell types, including duct epithelial, acinar, mesenchymal, and neuronal cells.18 Acinar cells secrete amylase into the digestive tract. The features resemble the pancreatic exocrine system. In the pancreas, a copper-deficient diet facilitates acinar cell depletion followed by progenitor cell proliferation. Obstruction of main excretory ducts is another model of tissue injury and regeneration of the pancreas.19 The same method is applied in the case of the salivary gland regarding tissue injury and regeneration.20 In the salivary gland, 1 week after ligation of the main excretory duct, almost all of the acinar cells disappeared as a result of apoptosis21,22 and extensive proliferation of the duct epithelium occurred.20 Reopening of the obstructed duct resulted in regeneration of normal gland tissues. During the regeneration process, the duct cells proliferate and differentiate into acinar cells within 5 days after the reopening.20 Intercalated duct cells may be precursors for both duct epithelial and acinar cells.23 These investigations suggested that the numbers of progenitors for acinar cells proliferate during tissue injury. We considered that the salivary gland might contain stem cells that can differentiate into cell types of other endodermal organs, such as the liver and pancreas. Transplantation of cells prepared from damaged salivary glands led to identification of epithelium-like cells that settled in the oval cell response area of the rat liver. We now report isolation and characterization of progenitor cells from the
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duct-ligated salivary gland. Our evidence shows that progenitor cells differentiate into both hepatic and pancreatic cell types.
Materials and Methods Isolation and Culture of Salivary Gland–Derived Cells. Inbred Crj:CD (Sprague-Dawley) rats and Crj: LEC (Long-Evans cinnamon) rats24,25 were purchased from Charles River Japan, Inc. (Osaka, Japan). LongEvans agouti (LEA) rats were a gift from Dr. K. Matsumoto (Institute for Animal Experimentation, University of Tokushima School of Medicine, Tokushima, Japan). All work was approved by the Center for Animal Resources and Development of Kumamoto University, and animals had humane care as outlined in the Guide for Care and Use of Laboratory Animals. Eight-week-old male LEA rats were anesthetized by giving inhaled ether and pentobarbital (30 mg/kg intraperitoneally; Nacalai Tesque, Inc., Kyoto, Japan) and then held in the supine position with extension of the front neck. The midline of the neck was incised, and both sides of the submandibular gland were exposed. After the glands had been inverted, main excretory ducts were double ligated; 6 days later, the glands were excised, minced, and incubated with 30 mL ethylene glycol-bis(-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid buffer2 at 37°C for 20 minutes and then centrifuged at 100g for 5 minutes at room temperature. Pellets were suspended in 60 mL digestion buffer containing Dulbecco’s modified Eagle medium/F12 1:1 (Invitrogen, Carlsbad, CA), 1.67 mg/mL collagenase (Invitrogen), and 1.33 mg/mL hyaluronidase (Nacalai Tesque, Inc.), and the suspension was incubated for 40 minutes at 37°C. The partly digested tissues were further treated with dispersion buffer containing Dulbecco’s modified Eagle medium/F12 and 1.67 mg/mL dispase (Invitrogen) and incubated for 60 minutes at 37°C. The cell suspensions were then passed through a stainless filter and centrifuged at 100g for 5 minutes at 4°C. The pellets were suspended in 10 mL Dulbecco’s modified Eagle medium/F12 1:1 medium and washed 3 times with Williams’ E medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). The isolated cells were plated at a density of 5 ⫻ 104 cells/100 mm in a type I collagencoated dish (Asahi Techno Glass, Tokyo, Japan) in Williams’ E medium supplemented with 10% fetal bovine serum, 20 ng/mL rat epidermal growth factor (Higeta Shoyu, Tokyo, Japan), 10⫺8 mol/L insulin (Invitrogen), 10⫺6 mol/L dexamethasone (Sigma Chemical Co., St. Louis, MO), 100 U/mL penicillin G, and 100 g/mL streptomycin (Invitrogen). Colonies were picked up using a cloning ring (Asahi Techno Glass). For a thick Matrigel
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(BD Biosciences Falcon, San Diego, CA) culture, the cells were cultured on 1:3 Matrigel diluted with serum-free Williams’ E medium. Analysis of Antigens in Cultured Cells. The immunostaining method was according to the manufacturer’s description. In brief, cultured cells on a glass-based dish (Asahi Techno Glass) were washed 3 times with phosphate-buffered saline, fixed in 4% paraformaldehyde (Nacalai Tesque, Inc.) for 20 minutes at 4°C, and then washed in phosphate-buffered saline including 0.2% Tween 20 (Wako Pure Chemical Inc., Osaka, Japan). Nonspecific binding was blocked with nonimmune serum of a species from which the secondary antibody had been obtained or nonspecific staining blocking reagent (DAKO A/S, Glostrup, Denmark). The primary antibodies used in this study were anti-laminin (DAKO), anti–type I collagen (LSL Co., Ltd., Tokyo, Japan), anti-␣61 integrin (Chemicon International, Inc., Temecula, CA), fluorescein isothiocyanate– conjugated anti-albumin (Inter-Cell Technologies, Inc., Hopewell, NJ), anti-AFP (DAKO), anti-insulin (Biogenesis Ltd., Poole, England), antiglucagon (DAKO), anti-rat nestin (BD Biosciences PharMingen, San Diego, CA), anti-III tubulin (Research Diagnostics, Inc., Flanders, NJ), anti– glial fibrillary acidic protein (DAKO), anti-amylase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti– cytokeratin 19 (Novocastra Laboratories Ltd., Newcastle, United Kingdom). The secondary antibodies used in this study were Alexa 488 –labeled anti-mouse immunoglobulin G or Alexa 594 –labeled anti-rabbit immunoglobulin G (Molecular Probes, Inc., Eugene, OR). Cells were viewed under an FV500 confocal laser scanning microscope (Olympus Optical Ltd., Tokyo, Japan). Cell staining and flow cytometric analysis were performed according to the manufacturer’s instructions. In brief, after 3 washings with staining buffer, sample cells were incubated with fluorescein isothiocyanate– conjugated anti-CD34 monoclonal antibody (Santa Cruz Biotechnology, Inc.), phycoerythrin-conjugated anti-CD44s monoclonal antibody (Immunotech, Marseille, France), biotinylated anti–Thy-1 monoclonal antibody (BD Biosciences PharMingen), anti-␣61integrin monoclonal antibody (Chemicon), or anti c-kit polyclonal antibody (Santa Cruz Biotechnology, Inc.). The secondary reagents were the phycoerythrin-streptavidin complex, Alexa 488 – conjugated anti-mouse immunoglobulin G, and Alexa 633– conjugated anti-rabbit immunoglobulin G. After 3 washings with staining buffer, labeled cells were analyzed using a FACS calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).
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Immunohistochemical Staining and In Situ Hybridization of Liver Specimens. Livers and salivary glands were fixed in 10% formaldehyde and embedded in paraffin. Sections (4 m thick) were incubated in 0.3% hydrogen peroxide in methanol for 30 minutes to inactivate internal peroxidase, washed with phosphate-buffered saline, and incubated with normal goat serum or normal sheep serum to block nonspecific binding of antibodies. The sections were then incubated with primary antibodies. The antibodies used were anti-rat albumin antibody (Inter-Cell Technologies), anti– cytokeratin 19 monoclonal antibody (Novocastra Laboratories), anti-laminin polyclonal antibody (DAKO), and anti-AFP polyclonal antibody (DAKO). To facilitate antigen retrieval, sections for the cytokeratin-19 immunostaining were digested with 0.05% trypsin (Sigma Chemical Co.) in 0.1 mol/L Tris-HCl and 0.1% CaCl2, pH 7.8, for 5 minutes at 37°C. Liver specimens obtained from a recipient rat that had undergone cell transplantation were subjected to in situ hybridization for the sry gene to detect donor (male)-derived cells. The sry probe was synthesized using polymerase chain reaction (PCR) DIG Labeling Mix (Roche, Basel, Switzerland). DIG-labeled PCR products were amplified using GeneAmp PCR 9700 (Perkin-Elmer Corp., Norwalk, CT) with a set of primers for sry26 (401 base pairs): forward, 5⬘-GTGTTCAGCCCTACAGCCTGA-3⬘; reverse, 5⬘-TTGTTGTCCCATTGCAGCAG-3⬘. GenPoint kits (DAKO) were used for the in situ hybridization according to the manufacturer’s instructions but with slight modifications. Transplantation of Cultured Cells. The 2-AAF pellet (time-release pellet, 2 mg/day; Innovative Research of America, Sarasota, FL) was inserted 7 days before twothirds hepatectomy according to Petersen et al.7 but with slight modifications. Nine-week-old female LEC rats, which served as recipients, were hepatectomized under anesthesia according to the methods described by Higgins and Anderson.27 Freshly isolated salivary gland cells (8 ⫻ 106 cells/rat) or cultured cells (1 ⫻ 106 cells/rat) were suspended with 200 L serum-free Williams’ E medium and injected into the inferior pole of the spleen of the recipient rat using a 29-gauge needle and syringe. Labeling of Cultured Cells. Recombinant retroviral vector expressing enhanced green fluorescent protein (EGFP) was prepared using pLEGFP-C1 (BD Biosciences Clontech, San Diego, CA) according to the manufacturer’s description. The SGP-1 cells were transduced and subjected to minimal dilution for selection of EGFPpositive cells. A single cell designated SGP-1/H2 that expressed EGFP was selected and expanded for further use.
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Fig. 1. Histologic changes in the salivary gland and isolated cells. (A-C) PAS staining of submandibular glands from a LEA rat (A) before and (B and C) 6 days after the duct ligation. (C) Ductal proliferation and disappearance of PAS-positive acinar cells occurred after main duct ligation. (D and E) Immunohistochemical staining for AFP in the submandibular gland. (D) Duct epithelial cells of normal salivary gland were weakly positive for AFP. (E) In contrast, after ligation of the main duct, AFP was not present in the duct epithelial cells. (F) Immunohistochemical staining of laminin in a section from a duct-ligated salivary gland. Lumens of proliferated small ducts are indicated by asterisks. In the periductal area, small cell clusters stained for laminin appeared. (G) A small colony formed by isolated epithelium-like cells on type I collagen in culture. (H) Small clusters formed by salivary gland– derived epithelial cells on type I collagen (arrows). (I) Clusters formed on type I collagen were stained with anti-laminin antibody. The extracellular matrix surrounding cell cluster was positive for laminin. (G-I) Scale bar ⫽ 50 m. (Original magnification: A and B, ⫻40; C and F, ⫻200; D and E, ⫻100.)
Reverse-Transcription PCR Analysis. For reversetranscription PCR analysis, total RNA was extracted from cultured cells and its clusters using ISOGEN kits (Nippon Gene, Tokyo, Japan). Complementary DNAs were prepared from 1 g of total RNA using Superscript II (Invitrogen) with oligo(dT) primer (Invitrogen) according to the manufacturer’s instructions. The resulting complementary DNA was amplified using GeneAmp PCR 9700 (PerkinElmer Corp.) with the following sets of primers.4,28,29 For albumin (436 base pairs): forward, 5⬘-ATACACCCAGAAAGCACCTC-3⬘; reverse, 5⬘-CACGAATTGTGCGAATGTCAC-3⬘; for AFP (686 base pairs): forward, 5⬘AACAGCAGAGTGCTGCAAAC-3⬘; reverse, 5⬘-AGGTTTCGTCCCTCAGAAAG-3⬘; for insulin (187 base pairs): forward, 5⬘-TGCCCAGGCTTTTGTCAAACAGCACCTT-3⬘; reverse, 5⬘-CTCCAGTGCCAAGGTCTGAA-3⬘; for glucagon (236 base pairs): forward, 5⬘-GTGGCTGGATTGTTTGTAATGCTG-3⬘; reverse, 5⬘-CGGTTCCTCTTGGTGTTCATCAAC-3⬘; for glyceraldehyde-3-phosphate dehydrogenase (580 base pairs): forward, 5⬘-ATCACTGCCACTCAGAAGAC-3⬘; reverse, 5⬘-TGAGGGAGATGCTCAGTGTT-3⬘; for Pdx-1 (305 base pairs): forward, 5⬘-
CGGCCACACAGCTCTACAAGG-3⬘; reverse, 5⬘-TTCCAGGCCCCCAGTCTCGG-3⬘; for Pax-4 (214 base pairs): forward, 5⬘-TGGCTTTCTGTCCTTCTGTGAGG-3⬘; reverse, 5⬘-TCCAAGACTCCTGTGCGGTAGTAG-3⬘; for Pax-6 (545 base pairs): forward, 5⬘-AAGAGTGGCGACTCCAGAAGTTG-3⬘; reverse, 5⬘-ACCACACCTGTATCCTTGCTTCAGG-3⬘; for Nkx2.2 (188 base pairs): forward, 5⬘-CACGCAGGTCAAGATCTG-3⬘; reverse, 5⬘-TGCCCGCCTGGAAGGTGGCG-3⬘. PCR products were electrophoresed on a 4% NuSieve GTG agarose (FMC Bioproducts, Rockland, ME) and visualized using ethidium bromide (Nacalai Tesque, Inc.) staining.
Results Histologic Changes in the Salivary Gland After Duct Ligation. Rat submandibular glands are composed of periodic acid–Schiff (PAS)-positive acinar cells and PAS-negative duct epithelial cells (Fig. 1A). Six days after ligation of the main excretory duct, almost all of the acinar cells had disappeared and were replaced by PAS-negative and smaller epithelial cells (Fig. 1B and C). This change,
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known as ductal proliferation, was associated with interstitial fibrosis and thickening of the basement membrane (Fig. 1C).20 The ductal epithelium was positive for AFP before ligation (Fig. 1D), but the AFP-positive cells had mostly disappeared after the ligation (Fig. 1E). Small ductal cells, which became predominant in the ligated gland, were AFP negative. Laminin immunostaining showed that both proliferated ducts and the thickened basement membrane were negative. However, there were few foci of laminin-positive cells after the ligation, and these foci were seen in the periductal area (Fig. 1F) and were positive for ␣61 integrin (data not shown). Without ligation, laminin and ␣61 integrin were both negative in the adult rat submandibular gland, as reported elsewhere.30 Isolation of Salivary Gland Cells. We prepared salivary gland cells as described in Materials and Methods. The recipient rats were treated with 2-AAF and underwent two-thirds hepatectomy as described; the rats were then given 2-AAF for 2 weeks and the livers resected and investigated using in situ hybridization for sry and srypositive donor-derived cells detected in the oval cell response area (data not shown). These experiments indicated that progenitors of liver cells were present in the ligated salivary gland. We next attempted to isolate the possible progenitor cells for the hepatocytes from the ligated salivary glands. In this experiment, salivary glands of male LEA rats were used. Various cell types appeared on the dish, and fibroblast-like cells and large epitheliumlike cells were predominant. Colonies of the cells with a small and round epithelium-like shape became visible, and these colonies were picked up and plated on type I collagen-coated 24-well plates for further purification. These cells were repeatedly obtained from different rat salivary glands using the same protocol and had the same morphology and characteristics. Approximately 5 to 10 colonies were identified within 7 days after the plating of 5 ⫻ 104 salivary gland cells. Cells from these colonies were purified in single form with limiting dilution on type I collagen-coated 96-well plates. One of the single purified cells were designated as “salivary gland– derived progenitor” SGP-1 (Fig. 1G) and used for further experiments. Doubling time of the SGP-1 cells is approximately 72 hours. SGP-1 cells grew for at least 3 months after the initial purification and without changing shape and characteristics. The doubling time of the cells was decreased after 30 to 50 passages, and the cells eventually stopped proliferating. When the cells were cultured on type I collagen-coated dishes, they formed small clusters (Fig. 1H). The extracellular matrix around the clusters was positive for laminin, in contrast to the monolayer SGP-1 sheet around the clusters (Fig. 1I). All experiments were performed using cells at passages between 6 and 15.
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Characterization of Undifferentiated SGP-1. Cells seeded at a low density were positive for both ␣61 integrin and intracellular laminin staining (Fig. 2A-C). SGP-1 cells at the monolayer stage were not positive for AFP, albumin, insulin, glucagon, amylase, glial fibrillary acidic protein, and Tuj-1 (data not shown). SGP-1 cells were weakly positive for type I collagen (data not shown). Flow cytometric analysis of SGP-1 cells, shown in Fig. 3, demonstrated that the cells were positive for ␣61 integrin (Fig. 3C and D), negative for CD34, and low positive for CD44s (Fig. 3B and D). CD44s were transiently positive in epithelium of acini and ducts in the developing salivary gland, but positive cells in the adult salivary gland were not evident.31 A small number of SGP-1 cells were positive for c-kit and Thy-1 (Fig. 3B and C). These proteins are characteristic markers of hepatic oval cells.32 After confluency was achieved and cell density increased, small clusters of SGP-1 appeared. In the small clusters, the cells were highly positive for ␣61 integrin. The ratio of Thy-1–positive cells gradually increased after SGP-1 formed clusters (data not shown). SGP-1 Cells Differentiate Into Endodermal Lineage. When SGP-1 cells formed clusters containing 30 to 100 cells, they differentiated into endodermal lineage on type I collagen-coated dishes (Fig. 4) and 2 major types of clusters appeared: one consisting of hepatocyte-like cell type (hepatic cluster) and the other islet-like cell type of the pancreas (pancreatic cluster). The hepatic clusters mainly contained cells positive for AFP and/or albumin (Fig. 2D-F). The pancreatic clusters mainly contained cells positive for glucagon and/or insulin (Fig. 2G-I). In the hepatic clusters, cells with AFP usually located at the center and cells with albumin tended to present at the periphery of these clusters. The cells with albumin or AFP were rare in the pancreatic clusters. Clusters containing hepatocyte-like and islet-like cells are equally generated when cultured on type I collagencoated dishes. The ratios of the cells in the clusters positive for AFP, albumin, glucagon, and insulin were 21.6%, 19.5%, 11.1%, and 15.0%, respectively. More than 80% of the clusters had differentiated. Under these conditions of cell culture, amylase- or cytokeratin 19 –positive cells were rare (⬍5%). Cells positive for these 2 cell type markers, representing acinar and duct epithelial cells, are predominant in the adult salivary gland. On a thick Matrigel culture with hepatocyte growth factor, SGP-1 cells differentiate into cytokeratin 19 –positive cells and amylasepositive cells (data not shown). These investigations suggested that cluster formation of the cells was essential for differentiation of SGP-1 on type I collagen. When SGP-1 cells were cultured without an extracellular matrix coating, pro-
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Fig. 2. Immunofluorescent stainings of SGP-1 and endodermal lineage-committed cell containing cluster. Staining for laminin was performed (A) without Tween 20 treatment or (B) after Tween 20 treatment. (A-C) Nuclei were stained by 4⬘,6-diamidino-2-phenylindole. Insets for A and B show laminin staining without the 4⬘,6-diamidino2-phenylindole image. (C) Tween 20 –treated SGP-1 were stained (green, ␣61 integrin; red, laminin). (Inset) SGP-1 staining without Tween 20 treatment. Hepatic clusters formed by SGP-1 on (D) type I collagen or on (E and F) laminin were stained using an anti-rat albumin antibody (green; D-F) and antiAFP antibody (red; D-F). On laminincoated dishes, all cells in clusters were positive for hepatic differentiation marker, albumin, and/or AFP (E and F). Pancreatic clusters formed by SGP-1 on type I collagen were stained using an anti-insulin antibody (green; G and I) and an antiglucagon antibody (red; H and I). Scale bars: A-C, 20 m; D-I, 100 m.
liferation, cluster formation, and differentiation of the cells were impaired. When the SGP-1 cells were transferred to laminin-coated dishes at a density of 3 ⫻ 104 cells/60-mm dish and cultured for 10 days, the proliferated cells in the clusters became the hepatic cell types (Fig. 2E and F). More than 90% of SGP-1 cells were positive for albumin or AFP when cultured under these conditions. In contrast, the production of islet-like cell types was negligible. Reverse-transcription PCR analysis of messenger RNA obtained from the cultured cells on type I collagen-coated dishes showed that clusters of SGP-1 cells expressed genes for AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2.4,28,29 However, cells of monolayer sheets did not express any of these genes (Fig. 5). This observation indicates that cluster formation plays a pivotal role for SGP-1 cells to differentiate into endodermal lineage. Transplantation of SGP-1 Cells Into Liver. To test in vivo differentiation of SGP-1 cells, 1 ⫻ 106 of the cells cultured as monolayers were injected into the spleen of recipient 9-week-old female LEC rats treated according to the 2-AAF/two-thirds hepatectomy protocol as described in Materials and Methods. Donor-derived cells in recipient liver tissue were detected by in situ hybridization using sry probe. Donor-derived cells were found in hepatocytes
Fig. 3. Flow cytometric analysis of SGP-1 cells cultured on type I collagen. SGP-1 cell suspensions were incubated using the following antibodies: (A) isotype control, (B) fluorescein isothiocyanate– conjugated anti-CD34 antibody and phycoerythrin-conjugated anti– c-kit antibody, (C) anti-␣61 integrin antibody and phycoerythrin-conjugated anti–Thy-1 antibody, and (D) anti-␣61 integrin antibody and phycoerythrin-conjugated anti-CD44s antibody. 7-Aminoactinomycin D was used to discriminate living cells from dead cells, which were excluded from cytometric analysis.
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Discussion
Fig. 4. Schematic presentation for proliferation and cluster formation of SGP-1 on type I collagen-coated dish. (A) Low-density phase. SGP-1 were plated at a low density (4.0 ⫻ 105 cells/100-mm dish) on type I collagen-coated dishes. At this phase, SGP-1 were positive for intracellular laminin (Fig. 2B and C). (B) High-density phase. Six to seven days after plating, density was approximately 3.0 to 3.5 ⫻ 106 cells/100-mm dish. At this phase, clusters were not observed. (C) Formation of small cluster phase. At high density on type I collagen, SGP-1 started to form small cell clusters (Fig. 1H). There were 1.0 to 3.0 ⫻ 104 clusters formed in the 100-mm dish, and part of the cells began to differentiate. (D) Formation of large cluster phase. The SGP-1 continued to grow and formed large clusters. Differentiated cells were present only in the clusters (Fig. 2D-I). Dark cells indicate differentiated cells.
and bile duct epithelial cells 2 weeks after the transplantation (Fig. 6A and B). These cells could not be distinguished from surrounding cells. Albumin immunostaining showed that these hepatocyte-like cells were positive for albumin (Fig. 6C). Next, we labeled the SGP-1 cells with EGFP using a retrovirus vector containing EGFP complementary DNA. The labeled cells differentiated into hepatocytelike cells, islet-like cells, and neural cells in vitro as parental SGP-1 cells (data not shown). A total of 2 ⫻ 106 of SGP-1/H2 were transplanted into recipient female rats; 2 weeks later, EGFP-positive cells in the recipient liver were visible as seen under a fluorescent microscope (Fig. 6D). A portion of small lobes in the liver was surrounded by EGFP-positive cells. These cells positive for GFP were also positive for albumin immunostaining (Fig. 6E and F).
Based on transplantation studies, we identified epithelium-like cells as a possible progenitor for liver cells (SGP1). Characteristic features of undifferentiated SGP-1 are that it expresses ␣61 integrin and laminin, which are ligands for ␣61 integrin. It was shown that duct cells of the salivary gland are positive for AFP33; however, SGP-1 cells were negative for AFP. Oval cells, a tissue stem cell of the liver, are positive for AFP, albumin, and cytokeratin 19. In addition, oval cells express Thy-1, CD34, and ckit, which are markers of hematopoietic stem cells.32 In contrast, SGP-1 is negative for AFP, albumin, cytokeratin 19, and CD34. These findings indicated that SGP-1 is distinct from the hepatic oval cell. Hepatic colony-forming units in culture (H-CFU-C) is a progenitor cell for hepatic cell types obtained from fetal livers of mice.34,35 The H-CFU-C cell is negative for c-kit and CD45, positive for AFP and c-met, and low positive for CD49f.35 The property of SGP-1 differs from H-CFU-C because it is highly positive for CD49f. In addition, H-CFU-C produces cytokeratin 19 –positive cells when cultured on laminin-coated dishes; however, production of cytokeratin 19 –positive cells from SGP-1 on laminin-coated dishes or on type I collagen-coated dishes was rare. Reverse-transcription PCR analyses of messenger RNA from undifferentiated SGP-1 confirmed that the cells do not express AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2. Thus, SGP-1 does not express specific genes characteristic of differentiated hepatocytes or pan-
Fig. 5. Reverse-transcription PCR analysis of messenger RNA obtained from cultured SGP-1 cells. Total RNAs were isolated from monolayer and clusters of SGP-1. Complementary DNAs transcribed from the RNAs by reverse transcriptase were used as templates for PCR with primers for glyceraldehyde-3-phosphate dehydrogenase, AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2.
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Fig. 6. Transplantation of SGP-1 cells. Liver sections obtained from recipient rats on day 14 after cell transplantation according to the 2-AAF/two-thirds hepatectomy protocol are shown. (A-C) Transplanted SGP-1 cells were detected by in situ hybridization using sry probe. Arrows indicate sry-positive nuclei. SGP-1– derived cells were found as hepatocyte-like cells (arrows; A and C) or bile duct epithelium-like cells (arrows; B). Fifteen rats were used for these experiments, and it was shown that (A) sry-positive hepatocyte was approximately 5.7% ⫾ 3.2% and (B) sry-positive bile duct epithelium was approximately 1.5% ⫾ 2.0%. SGP-1– derived hepatocyte-like cells (arrows; C) were immunoreactive with anti-albumin antibody. (D) EGFP-labeled SGP-1 (SGP-1/H2) cells transplanted into the rat liver were detected 14 days after the transplantation using fluorescent microscopy (green; D). (E) Green fluorescent protein (GFP)-positive cells formed foci in the liver section. Five to 10 large foci were present in the liver, and many small foci had formed. Approximately 5% to 10% of the hepatocytes were GFP positive, as seen in 3 independent experiments. (F) GFP-positive cells were positive for albumin staining. Sections counterstained with (A-C) hematoxylin or (E and F) 4⬘,6-diamidino-2-phenylindole. (Original magnification: A-C, E, and F, ⫻400.)
creatic islet cells. These features are unique among previously described tissue stem cells for liver1,32,34,35 and pancreas.11,36 SGP-1 can be maintained in culture on type I collagencoated dishes for at least 3 months without losing the capacity to generate diverse cell types in culture. The cells are sensitive for irradiation-induced cell arrest and do not form a teratoma or a transformed tumor when transplanted subcutaneously into SCID mice. Single cell cultures of SGP-1 repeatedly produced original multipotent
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progenitor cells capable of differentiation into various cell types. However, once the cells are differentiated, they do not produce multipotent progenitor cells; therefore, undifferentiated cells are necessary to produce new progenitors. These results suggested that continuous renewal of multipotent SGP-1 should occur during culture on type I collagen. SGP-1 becomes hepatic cell types when transplanted via the portal vein. We performed transplantation of freshly isolated salivary gland cells or cultured SGP-1 using 2 different methods. First, we detected the donor cells by means of in situ hybridization. This procedure was used for transplantation of freshly isolated cells from the salivary gland, and we found that small numbers of the transplanted cells are present in the area of oval cell responses. Similarly, some of the SGP-1 cells are present in the oval cell response area after the transplantation. These cells could not be distinguished from the surrounding oval cell. It is possible that some of the progenitor cells in the ligated salivary glands may follow the process of differentiation of hepatic stem cells present in the liver; however, further experiments will be needed to clarify this possibility. SGP-1 is likely to be capable of differentiating into hepatic cell types when transplanted into the liver. Alternatively, the transplantation experiments using EGFP-labeled cells demonstrated that the transplanted cells show focal expansion, suggesting that SGP-1 is capable of multiplying in the liver. These results suggested that SGP-1 has the capacity of a precursor cell for hepatic cell types. The efficacy of retroviral transduction is as high as cultured fibroblasts; hence, SGP-1 may be a suitable target for gene therapy using a retrovirus vector. Differentiation of SGP-1 to hepatic cell types was achieved by culturing the cells on type I collagen-coated dishes or on laminin-coated dishes. The SGP-1 express AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2 after the formation of clusters on type I collagen-coated dishes. Pdx-1 is one of the early genes functioning during development of the pancreas. Pax-4 and Pax-6 are homeobox genes, and expression of these genes is essential for maturation of islets. These results suggested that expression of insulin and glucagon genes are consequences of sequential activation of genes that participate in the development of islets. Further characterization of the differentiation process may clarify the precise step involved in the production of hepatic cell type form SGP-1. In this context, SGP-1 will be useful to investigate genes involved in early development of the mammalian liver. Cluster formation is essential for differentiation of SGP-1 cells in culture. In hepatic oval cells, the formation of cell clusters led to differentiation into pancreatic endo-
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crine cells when cultured in medium containing 10% fetal calf serum and 23 mmol/L glucose.4 Cell-cell interactions should play an important role in differentiation processes in stem/progenitor cells. Our experimental data indicated that the presence of extracellular matrix laminin led the SGP-1 to the hepatic cell type. Because ␣61 integrin is expressed in SGP-1, laminin should be one of the important proteins that affects the fate of SGP-1. Alternatively, on a Matrigel culture with hepatocyte growth factor, SGP-1 cells produced cytokeratin 19 –positive cells and amylase-positive cells. There is evidence to support the notion that cell fates of tissue stem cells largely depend on the extracellular matrix.37,38 SGP-1 expresses laminin. Small numbers of lamininpositive cells appeared in the ligated glands, and these cells were not seen in the nonligated glands. Laminin-positive cells are seen in the terminal clusters during development of the salivary gland.30,39,40 Another characteristic feature of SGP-1 is that the cells are low positive for CD44s. CD44s is expressed during salivary gland morphogenesis (embryonic day 16 to birth in rats), and expression of CD44s was observed in the developing pancreas. However, it is not expressed during development of the liver.31 CD44s is a receptor for hyaluronate, has a binding domain for fibronectin, glycosaminoglycans, and is involved in cell-cell interaction. These findings in the SGP-1 suggested that this cell has some immature features of salivary gland cells. Bone marrow– derived stem cells can circulate and distribute in peripheral tissues and contribute to tissue regeneration.28,41,42 It is possible that SGP-1 cells are derived from bone marrow, and we tested this possibility by bone marrow transplantation to wild-type mice using ROSA26 mice carrying the -galactosidase gene as transgenic gene donors. These experiments showed that cells proliferating in the salivary gland after ligation were not derived from donor ROSA26 mice (Hisatomi et al., manuscript in preparation). Thus, SGP-1 is likely to originate from salivary gland tissue. Clinical use of adult progenitor cells is of great interest. Isolation of tissue stem cells from accessible organs and efficient induction of differentiation into specific tissue is indispensable for clinical application for cell transplantation therapy. We showed that in vitro culture of SGP-1 on laminin successfully induced hepatocyte-like cells producing AFP and albumin. These progenitor cells from salivary glands may prove suitable for liver cell transplantation.
References 1. Golding M, Sarraf CE, Lalani EN, Anilkumar TV, Edwards RJ, Nagy P, Thorgeirsson SS, et al. Oval cell differentiation into hepatocytes in the acetylaminofluorene-treated regenerating rat liver. HEPATOLOGY 1995;22: 1243-1253.
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2. Yasui O, Miura N, Terada K, Kawarada Y, Koyama K, Sugiyama T. Isolation of oval cells from Long-Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver. HEPATOLOGY 1997;25:329334. 3. Yang L, Faris RA, Hixson DC. Phenotypic heterogeneity within clonogenic ductal cell populations isolated from normal adult rat liver. Proc Soc Exp Biol Med 1993;204:280-288. 4. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99:80788083. 5. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276:60-66. 6. Grompe M, Finegold MJ. Liver stem cells. In: Marshak DR, Gardner RL, Gottlieb D, eds. Stem Cell Biology. New York: Cold Spring Harbor Laboratory, 2001:471-472. 7. Petersen BE, Zajac VF, Michalopoulos GK. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats. HEPATOLOGY 1998;27:1030-1038. 8. Dabeva MD, Hwang SG, Vasa SR, Hurston E, Novikoff PM, Hixson DC, Gupta S, et al. Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver. Proc Natl Acad Sci U S A 1997;94:7356-7361. 9. Rao MS, Dwivedi RS, Yeldandi AV, Subbarao V, Tan XD, Usman MI, Thangada S, et al. Role of periductal and ductular epithelial cells of the adult rat pancreas in pancreatic hepatocyte lineage. A change in the differentiation commitment. Am J Pathol 1989;134:1069-1086. 10. Rao MS, Subbarao V, Sato K, Reddy JK. Alterations of pancreatic hepatocytes in rats exposed to carcinogens. Am J Pathol 1991;139:1111-1117. 11. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001;50:521-533. 12. Shen CN, Slack JM, Tosh D. Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2000;2:879-887. 13. Tosh D, Shen CN, Slack JM. Differentiated properties of hepatocytes induced from pancreatic cells. HEPATOLOGY 2002;36:534-543. 14. Wang X, Al-Dhalimy M, Lagasse E, Finegold M, Grompe M. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 2001;158:571-579. 15. Bisgaard HC, Thorgeirsson SS. Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas. J Cell Physiol 1991;147:333-343. 16. Deutsch G, Jung J, Zheng M, Lora J, Zaret KS. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 2001;128:871-881. 17. Larson, WJ. Human Embryology. 3rd ed. Edinburgh: Churchill Livingstone, 2001. 18. Fawcett DW. A Textbook of Histology. 11th ed. Philadelphia: Saunders, 1986:587-597. 19. Abe K, Watanabe S. Apoptosis of mouse pancreatic acinar cells after duct ligation. Arch Histol Cytol 1995;58:221-229. 20. Takahashi S, Schoch E, Walker NI. Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction. Int J Exp Pathol 1998;79:293-301. 21. Walker NI, Gobe GC. Cell death and cell proliferation during atrophy of the rat parotid gland induced by duct obstruction. J Pathol 1987;153:333344. 22. Walker NI. Ultrastructure of the rat pancreas after experimental duct ligation. I. The role of apoptosis and intraepithelial macrophages in acinar cell deletion. Am J Pathol 1987;126:439-451. 23. Man YG, Ball WD, Marchetti L, Hand AR. Contributions of intercalated duct cells to the normal parenchyma of submandibular glands of adult rats. Anat Rec 2001;263:202-214. 24. Nakamura K, Endo F, Ueno T, Awata H, Tanoue A, Matsuda I. Excess copper and ceruloplasmin biosynthesis in long-term cultured hepatocytes
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from Long-Evans Cinnamon (LEC) rats, a model of Wilson disease. J Biol Chem 1995;270:7656-7660. Nagano K, Nakamura K, Urakami KI, Umeyama K, Uchiyama H, Koiwai K, Hattori S, et al. Intracellular distribution of the Wilson’s disease gene product (ATPase7B) after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat, an animal model of Wilson’s disease. HEPATOLOGY 1998;27:799-807. An J, Beauchemin N, Albanese J, Abney TO, Sullivan AK. Use of a rat cDNA probe specific for the Y chromosome to detect male-derived cells. J Androl 1997;18:289-293. Higgins GM, Anderson RM. Experimental pathology of the liver: I. Restoration of the liver of the white rat following surgical removal. Arch Pathol 1931;12:186-202. Oh SH, Miyazaki M, Kouchi H, Inoue Y, Sakaguchi M, Tsuji T, Shima N, et al. Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun 2000;279:500-504. Zhou J, Wang X, Pineyro MA, Egan JM. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 1999;48:2358-2366. Lazowski KW, Mertz PM, Redman RS, Kousvelari E. Temporal and spatial expression of laminin, collagen types IV and I and alpha 6/beta 1 integrin receptor in the developing rat parotid gland. Differentiation 1994;56:75-82. Weber B, Rosel M, Arch R, Moller P, Zoller M. Transient expression of CD44 variant isoforms in the ontogeny of the rat: Ectoderm-, endodermand mesoderm-derived cells express different exon combinations. Differentiation 1996;60:17-29. Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. HEPATOLOGY 1998;27:433-445. Tsuji T, Nagai N. Production of alpha-fetoprotein by human submandibular gland. Int J Dev Biol 1993;37:497-498.
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34. Suzuki A, Zheng YW, Kondo R, Kusakabe M, Takeda Y, Fukao K, Nakauchi H, et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. HEPATOLOGY 2000;32: 1230-1239. 35. Suzuki A, Zheng YW, Kaneko S, Onodera M, Fukao K, Nakauchi H, Taniguchi H. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 2002;156:173184. 36. Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 2002;143:3152-3161. 37. Jiang FX, Cram DS, DeAizpurua HJ, Harrison LC. Laminin-1 promotes differentiation of fetal mouse pancreatic beta-cells. Diabetes 1999;48:722730. 38. Brill S, Zvibel I, Halpern Z, Oren R. The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes. Eur J Cell Biol 2002;81:43-50. 39. Kadoya Y, Yamashina S. Intracellular accumulation of basement membrane components during morphogenesis of rat submandibular gland. J Histochem Cytochem 1989;37:1387-1392. 40. Kadoya Y, Kadoya K, Durbeej M, Holmvall K, Sorokin L, Ekblom P. Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes J Cell Biol 1995;129:521-534. 41. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, et al. Purified hematopoietic stem cells can differentiate into hepatocyte in vivo. Nat Med 2000;6:1229-1234. 42. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168-1170.
Salivary Gland Progenitor Cells Induced by Duct Ligation Differentiate Into Hepatic and Pancreatic Lineages Kenji Okumura,1 Kimitoshi Nakamura,1 Yuichiro Hisatomi,1 Koji Nagano,1 Yasuhiko Tanaka,1 Kunihiko Terada,2 Toshihiro Sugiyama,2 Kazuhiro Umeyama,3 Kozo Matsumoto,4 Tetsuro Yamamoto,5 and Fumio Endo1 Tissue damage can be assessed based on regenerative responses, including progenitor cell proliferation. In the salivary gland, tissue damage induced by ligation of main ducts leads to the disappearance of acinar cells and to marked proliferation of ductal cells. Reopening of the ducts leads to repopulation of acinar cells within 1 to 2 weeks, which suggests activation of tissue progenitor cells in a damaged state. Because submandibular glands derive from the endoderm and ectoderm, we investigated the possibility of the presence of endodermal progenitor cells. We cultured cells obtained from the ligated salivary gland and identified colonies of epithelium-like cells. We singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The SGP-1 expresses both ␣61 integrin and cytoplasmic laminin. The hematopoietic stem cell marker CD34 and hepatic oval cell markers such as albumin, ␣-fetoprotein (AFP), and cytokeratin 19 are all negative. However, when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into hepatic trabecula and produced albumin. When SGP-1 cells formed clusters on type I collagen-coated dishes, they differentiated into endodermal lineage and 2 major types of clusters appeared: one contained cells positive for AFP and/or albumin (hepatic cluster) and the other positive for glucagon and/or insulin (pancreatic cluster). On laminin-coated dishes, SGP-1 selectively differentiated into hepatic-type cells. In conclusion, the multipotent progenitor cells isolated from the rat salivary gland have characteristics of tissue stem cells and can differentiate into cells of endodermal lineages. (HEPATOLOGY 2003;38:104-113.)
T
he oval cell, a tissue stem cell for liver cell types, can differentiate into hepatocytes and bile duct epithelium when transplanted into the liver.1,2 These cells are a heterogeneous population positive for both hepatocyte and bile ductal cell markers such as albuAbbreviations: AFP, ␣-fetoprotein; 2-AAF, 2-acetylaminofluorene; LEA, LongEvans agouti; PCR, polymerase chain reaction; EGFP, enhanced green fluorescent protein; PAS, periodic acid–Schiff; H-CFU-C, hepatic colony-forming units in culture. From the 1Department of Pediatrics, School of Medicine, and the 5Division of Molecular Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2Department of Biochemistry, Akita University School of Medicine, Akita, Japan; 3Department of Cell Biology, Bios Inc., Kamakura, Japan; and 4Institute for Animal Experimentation, University of Tokushima School of Medicine, Tokushima, Japan. Received February 13, 2003; accepted March 29, 2003. Supported in part by grants for Research for the Future Program of JSPS, grantin-aid for Scientific Research and grant-in-aid for 21st Century COE Research from the Ministry of Education, Science, Technology, Sports and Culture, “Cell Fate Regulation Research and Education Unit,” and a research grant from the Ministry of Health, Labour and Welfare. Address reprint requests to: Fumio Endo, M.D., Ph.D., Department of Pediatrics, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 8608556, Japan. E-mail: [email protected]; fax: (81) 96-366-3471. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3801-0015$30.00/0 doi:10.1053/jhep.2003.50259 104
min, ␣-fetoprotein (AFP), cytokeratin 19, and other bile duct antigens.3 Oval cell populations also contain hematopoietic cell surface antigen–positive cells. The Thy-1 highly positive oval cell can differentiate into pancreatic cell types that secrete insulin, glucagon, and pancreatic polypeptide when cultured in the presence of high concentrations (23 mmol/L) of glucose.4 In the liver, after two-thirds hepatectomy, the regeneration process rapidly restores the original mass of the liver.5 During this process, oval cells do not appear and regeneration of the liver after hepatectomy is not likely to depend on tissue stem cells. In contrast, oval cells are seen during the process of progenitor-dependent liver regeneration.6 Progenitor-dependent liver regeneration has been shown under several experimental conditions. Administration of 2-acetylaminofluorene (2-AAF) combined with partial hepatectomy is one representative protocol.7 Under these conditions, 2-AAF suppresses hepatocyte proliferation and oval cells appear in the periportal area before restoration. These observations on hepatic oval cells imply that multipotent progenitor cells are induced during tissue injury in various organs. The pancreas is an organ of endodermal origin, and tissue injury can lead to the appearance of multipotent
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stem cells for the liver and the pancreas. When weaning rats are fed a copper-deficient diet for 8 weeks, these manipulations result in a marked reduction in the acinar mass and cytokeratin 19 –positive duct-like epithelial cells proliferate.8 If they are refed copper, cells with multiple hepatocellular characteristics emerge from the remaining pancreatic ducts.9 This work has been interpreted to suggest the presence of a pancreatic liver stem cell,10 and Dabeva et al. reported that pancreatic epithelial progenitor cells isolated from the copper-deficient diet rat pancreas can differentiate into hepatocytes when transplanted into the liver.8 Recent studies have shown that there is a tissue stem cell in the pancreas that can differentiate into hepatocytes in addition to pancreatic cell types. Zulewski et al. reported that multipotent stem cells from rat and human islets differentiated into hepatic cell types in culture when the cell density increased.11 In other experiments, a cell line from pancreatic acinar cells differentiated into hepatocytes in the presence of dexamethasone.12,13 In addition, cells including the pancreatic duct area repopulated in the liver.14 Thus, tissue stem cells isolated from liver and pancreas can undergo trans-differentiation to cells of other tissues.15,16 The salivary gland is derived from the endoderm, and the ectoderm participates in organogenesis.17 Salivary glands consist of many cell types, including duct epithelial, acinar, mesenchymal, and neuronal cells.18 Acinar cells secrete amylase into the digestive tract. The features resemble the pancreatic exocrine system. In the pancreas, a copper-deficient diet facilitates acinar cell depletion followed by progenitor cell proliferation. Obstruction of main excretory ducts is another model of tissue injury and regeneration of the pancreas.19 The same method is applied in the case of the salivary gland regarding tissue injury and regeneration.20 In the salivary gland, 1 week after ligation of the main excretory duct, almost all of the acinar cells disappeared as a result of apoptosis21,22 and extensive proliferation of the duct epithelium occurred.20 Reopening of the obstructed duct resulted in regeneration of normal gland tissues. During the regeneration process, the duct cells proliferate and differentiate into acinar cells within 5 days after the reopening.20 Intercalated duct cells may be precursors for both duct epithelial and acinar cells.23 These investigations suggested that the numbers of progenitors for acinar cells proliferate during tissue injury. We considered that the salivary gland might contain stem cells that can differentiate into cell types of other endodermal organs, such as the liver and pancreas. Transplantation of cells prepared from damaged salivary glands led to identification of epithelium-like cells that settled in the oval cell response area of the rat liver. We now report isolation and characterization of progenitor cells from the
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duct-ligated salivary gland. Our evidence shows that progenitor cells differentiate into both hepatic and pancreatic cell types.
Materials and Methods Isolation and Culture of Salivary Gland–Derived Cells. Inbred Crj:CD (Sprague-Dawley) rats and Crj: LEC (Long-Evans cinnamon) rats24,25 were purchased from Charles River Japan, Inc. (Osaka, Japan). LongEvans agouti (LEA) rats were a gift from Dr. K. Matsumoto (Institute for Animal Experimentation, University of Tokushima School of Medicine, Tokushima, Japan). All work was approved by the Center for Animal Resources and Development of Kumamoto University, and animals had humane care as outlined in the Guide for Care and Use of Laboratory Animals. Eight-week-old male LEA rats were anesthetized by giving inhaled ether and pentobarbital (30 mg/kg intraperitoneally; Nacalai Tesque, Inc., Kyoto, Japan) and then held in the supine position with extension of the front neck. The midline of the neck was incised, and both sides of the submandibular gland were exposed. After the glands had been inverted, main excretory ducts were double ligated; 6 days later, the glands were excised, minced, and incubated with 30 mL ethylene glycol-bis(-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid buffer2 at 37°C for 20 minutes and then centrifuged at 100g for 5 minutes at room temperature. Pellets were suspended in 60 mL digestion buffer containing Dulbecco’s modified Eagle medium/F12 1:1 (Invitrogen, Carlsbad, CA), 1.67 mg/mL collagenase (Invitrogen), and 1.33 mg/mL hyaluronidase (Nacalai Tesque, Inc.), and the suspension was incubated for 40 minutes at 37°C. The partly digested tissues were further treated with dispersion buffer containing Dulbecco’s modified Eagle medium/F12 and 1.67 mg/mL dispase (Invitrogen) and incubated for 60 minutes at 37°C. The cell suspensions were then passed through a stainless filter and centrifuged at 100g for 5 minutes at 4°C. The pellets were suspended in 10 mL Dulbecco’s modified Eagle medium/F12 1:1 medium and washed 3 times with Williams’ E medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). The isolated cells were plated at a density of 5 ⫻ 104 cells/100 mm in a type I collagencoated dish (Asahi Techno Glass, Tokyo, Japan) in Williams’ E medium supplemented with 10% fetal bovine serum, 20 ng/mL rat epidermal growth factor (Higeta Shoyu, Tokyo, Japan), 10⫺8 mol/L insulin (Invitrogen), 10⫺6 mol/L dexamethasone (Sigma Chemical Co., St. Louis, MO), 100 U/mL penicillin G, and 100 g/mL streptomycin (Invitrogen). Colonies were picked up using a cloning ring (Asahi Techno Glass). For a thick Matrigel
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(BD Biosciences Falcon, San Diego, CA) culture, the cells were cultured on 1:3 Matrigel diluted with serum-free Williams’ E medium. Analysis of Antigens in Cultured Cells. The immunostaining method was according to the manufacturer’s description. In brief, cultured cells on a glass-based dish (Asahi Techno Glass) were washed 3 times with phosphate-buffered saline, fixed in 4% paraformaldehyde (Nacalai Tesque, Inc.) for 20 minutes at 4°C, and then washed in phosphate-buffered saline including 0.2% Tween 20 (Wako Pure Chemical Inc., Osaka, Japan). Nonspecific binding was blocked with nonimmune serum of a species from which the secondary antibody had been obtained or nonspecific staining blocking reagent (DAKO A/S, Glostrup, Denmark). The primary antibodies used in this study were anti-laminin (DAKO), anti–type I collagen (LSL Co., Ltd., Tokyo, Japan), anti-␣61 integrin (Chemicon International, Inc., Temecula, CA), fluorescein isothiocyanate– conjugated anti-albumin (Inter-Cell Technologies, Inc., Hopewell, NJ), anti-AFP (DAKO), anti-insulin (Biogenesis Ltd., Poole, England), antiglucagon (DAKO), anti-rat nestin (BD Biosciences PharMingen, San Diego, CA), anti-III tubulin (Research Diagnostics, Inc., Flanders, NJ), anti– glial fibrillary acidic protein (DAKO), anti-amylase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti– cytokeratin 19 (Novocastra Laboratories Ltd., Newcastle, United Kingdom). The secondary antibodies used in this study were Alexa 488 –labeled anti-mouse immunoglobulin G or Alexa 594 –labeled anti-rabbit immunoglobulin G (Molecular Probes, Inc., Eugene, OR). Cells were viewed under an FV500 confocal laser scanning microscope (Olympus Optical Ltd., Tokyo, Japan). Cell staining and flow cytometric analysis were performed according to the manufacturer’s instructions. In brief, after 3 washings with staining buffer, sample cells were incubated with fluorescein isothiocyanate– conjugated anti-CD34 monoclonal antibody (Santa Cruz Biotechnology, Inc.), phycoerythrin-conjugated anti-CD44s monoclonal antibody (Immunotech, Marseille, France), biotinylated anti–Thy-1 monoclonal antibody (BD Biosciences PharMingen), anti-␣61integrin monoclonal antibody (Chemicon), or anti c-kit polyclonal antibody (Santa Cruz Biotechnology, Inc.). The secondary reagents were the phycoerythrin-streptavidin complex, Alexa 488 – conjugated anti-mouse immunoglobulin G, and Alexa 633– conjugated anti-rabbit immunoglobulin G. After 3 washings with staining buffer, labeled cells were analyzed using a FACS calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).
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Immunohistochemical Staining and In Situ Hybridization of Liver Specimens. Livers and salivary glands were fixed in 10% formaldehyde and embedded in paraffin. Sections (4 m thick) were incubated in 0.3% hydrogen peroxide in methanol for 30 minutes to inactivate internal peroxidase, washed with phosphate-buffered saline, and incubated with normal goat serum or normal sheep serum to block nonspecific binding of antibodies. The sections were then incubated with primary antibodies. The antibodies used were anti-rat albumin antibody (Inter-Cell Technologies), anti– cytokeratin 19 monoclonal antibody (Novocastra Laboratories), anti-laminin polyclonal antibody (DAKO), and anti-AFP polyclonal antibody (DAKO). To facilitate antigen retrieval, sections for the cytokeratin-19 immunostaining were digested with 0.05% trypsin (Sigma Chemical Co.) in 0.1 mol/L Tris-HCl and 0.1% CaCl2, pH 7.8, for 5 minutes at 37°C. Liver specimens obtained from a recipient rat that had undergone cell transplantation were subjected to in situ hybridization for the sry gene to detect donor (male)-derived cells. The sry probe was synthesized using polymerase chain reaction (PCR) DIG Labeling Mix (Roche, Basel, Switzerland). DIG-labeled PCR products were amplified using GeneAmp PCR 9700 (Perkin-Elmer Corp., Norwalk, CT) with a set of primers for sry26 (401 base pairs): forward, 5⬘-GTGTTCAGCCCTACAGCCTGA-3⬘; reverse, 5⬘-TTGTTGTCCCATTGCAGCAG-3⬘. GenPoint kits (DAKO) were used for the in situ hybridization according to the manufacturer’s instructions but with slight modifications. Transplantation of Cultured Cells. The 2-AAF pellet (time-release pellet, 2 mg/day; Innovative Research of America, Sarasota, FL) was inserted 7 days before twothirds hepatectomy according to Petersen et al.7 but with slight modifications. Nine-week-old female LEC rats, which served as recipients, were hepatectomized under anesthesia according to the methods described by Higgins and Anderson.27 Freshly isolated salivary gland cells (8 ⫻ 106 cells/rat) or cultured cells (1 ⫻ 106 cells/rat) were suspended with 200 L serum-free Williams’ E medium and injected into the inferior pole of the spleen of the recipient rat using a 29-gauge needle and syringe. Labeling of Cultured Cells. Recombinant retroviral vector expressing enhanced green fluorescent protein (EGFP) was prepared using pLEGFP-C1 (BD Biosciences Clontech, San Diego, CA) according to the manufacturer’s description. The SGP-1 cells were transduced and subjected to minimal dilution for selection of EGFPpositive cells. A single cell designated SGP-1/H2 that expressed EGFP was selected and expanded for further use.
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Fig. 1. Histologic changes in the salivary gland and isolated cells. (A-C) PAS staining of submandibular glands from a LEA rat (A) before and (B and C) 6 days after the duct ligation. (C) Ductal proliferation and disappearance of PAS-positive acinar cells occurred after main duct ligation. (D and E) Immunohistochemical staining for AFP in the submandibular gland. (D) Duct epithelial cells of normal salivary gland were weakly positive for AFP. (E) In contrast, after ligation of the main duct, AFP was not present in the duct epithelial cells. (F) Immunohistochemical staining of laminin in a section from a duct-ligated salivary gland. Lumens of proliferated small ducts are indicated by asterisks. In the periductal area, small cell clusters stained for laminin appeared. (G) A small colony formed by isolated epithelium-like cells on type I collagen in culture. (H) Small clusters formed by salivary gland– derived epithelial cells on type I collagen (arrows). (I) Clusters formed on type I collagen were stained with anti-laminin antibody. The extracellular matrix surrounding cell cluster was positive for laminin. (G-I) Scale bar ⫽ 50 m. (Original magnification: A and B, ⫻40; C and F, ⫻200; D and E, ⫻100.)
Reverse-Transcription PCR Analysis. For reversetranscription PCR analysis, total RNA was extracted from cultured cells and its clusters using ISOGEN kits (Nippon Gene, Tokyo, Japan). Complementary DNAs were prepared from 1 g of total RNA using Superscript II (Invitrogen) with oligo(dT) primer (Invitrogen) according to the manufacturer’s instructions. The resulting complementary DNA was amplified using GeneAmp PCR 9700 (PerkinElmer Corp.) with the following sets of primers.4,28,29 For albumin (436 base pairs): forward, 5⬘-ATACACCCAGAAAGCACCTC-3⬘; reverse, 5⬘-CACGAATTGTGCGAATGTCAC-3⬘; for AFP (686 base pairs): forward, 5⬘AACAGCAGAGTGCTGCAAAC-3⬘; reverse, 5⬘-AGGTTTCGTCCCTCAGAAAG-3⬘; for insulin (187 base pairs): forward, 5⬘-TGCCCAGGCTTTTGTCAAACAGCACCTT-3⬘; reverse, 5⬘-CTCCAGTGCCAAGGTCTGAA-3⬘; for glucagon (236 base pairs): forward, 5⬘-GTGGCTGGATTGTTTGTAATGCTG-3⬘; reverse, 5⬘-CGGTTCCTCTTGGTGTTCATCAAC-3⬘; for glyceraldehyde-3-phosphate dehydrogenase (580 base pairs): forward, 5⬘-ATCACTGCCACTCAGAAGAC-3⬘; reverse, 5⬘-TGAGGGAGATGCTCAGTGTT-3⬘; for Pdx-1 (305 base pairs): forward, 5⬘-
CGGCCACACAGCTCTACAAGG-3⬘; reverse, 5⬘-TTCCAGGCCCCCAGTCTCGG-3⬘; for Pax-4 (214 base pairs): forward, 5⬘-TGGCTTTCTGTCCTTCTGTGAGG-3⬘; reverse, 5⬘-TCCAAGACTCCTGTGCGGTAGTAG-3⬘; for Pax-6 (545 base pairs): forward, 5⬘-AAGAGTGGCGACTCCAGAAGTTG-3⬘; reverse, 5⬘-ACCACACCTGTATCCTTGCTTCAGG-3⬘; for Nkx2.2 (188 base pairs): forward, 5⬘-CACGCAGGTCAAGATCTG-3⬘; reverse, 5⬘-TGCCCGCCTGGAAGGTGGCG-3⬘. PCR products were electrophoresed on a 4% NuSieve GTG agarose (FMC Bioproducts, Rockland, ME) and visualized using ethidium bromide (Nacalai Tesque, Inc.) staining.
Results Histologic Changes in the Salivary Gland After Duct Ligation. Rat submandibular glands are composed of periodic acid–Schiff (PAS)-positive acinar cells and PAS-negative duct epithelial cells (Fig. 1A). Six days after ligation of the main excretory duct, almost all of the acinar cells had disappeared and were replaced by PAS-negative and smaller epithelial cells (Fig. 1B and C). This change,
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known as ductal proliferation, was associated with interstitial fibrosis and thickening of the basement membrane (Fig. 1C).20 The ductal epithelium was positive for AFP before ligation (Fig. 1D), but the AFP-positive cells had mostly disappeared after the ligation (Fig. 1E). Small ductal cells, which became predominant in the ligated gland, were AFP negative. Laminin immunostaining showed that both proliferated ducts and the thickened basement membrane were negative. However, there were few foci of laminin-positive cells after the ligation, and these foci were seen in the periductal area (Fig. 1F) and were positive for ␣61 integrin (data not shown). Without ligation, laminin and ␣61 integrin were both negative in the adult rat submandibular gland, as reported elsewhere.30 Isolation of Salivary Gland Cells. We prepared salivary gland cells as described in Materials and Methods. The recipient rats were treated with 2-AAF and underwent two-thirds hepatectomy as described; the rats were then given 2-AAF for 2 weeks and the livers resected and investigated using in situ hybridization for sry and srypositive donor-derived cells detected in the oval cell response area (data not shown). These experiments indicated that progenitors of liver cells were present in the ligated salivary gland. We next attempted to isolate the possible progenitor cells for the hepatocytes from the ligated salivary glands. In this experiment, salivary glands of male LEA rats were used. Various cell types appeared on the dish, and fibroblast-like cells and large epitheliumlike cells were predominant. Colonies of the cells with a small and round epithelium-like shape became visible, and these colonies were picked up and plated on type I collagen-coated 24-well plates for further purification. These cells were repeatedly obtained from different rat salivary glands using the same protocol and had the same morphology and characteristics. Approximately 5 to 10 colonies were identified within 7 days after the plating of 5 ⫻ 104 salivary gland cells. Cells from these colonies were purified in single form with limiting dilution on type I collagen-coated 96-well plates. One of the single purified cells were designated as “salivary gland– derived progenitor” SGP-1 (Fig. 1G) and used for further experiments. Doubling time of the SGP-1 cells is approximately 72 hours. SGP-1 cells grew for at least 3 months after the initial purification and without changing shape and characteristics. The doubling time of the cells was decreased after 30 to 50 passages, and the cells eventually stopped proliferating. When the cells were cultured on type I collagen-coated dishes, they formed small clusters (Fig. 1H). The extracellular matrix around the clusters was positive for laminin, in contrast to the monolayer SGP-1 sheet around the clusters (Fig. 1I). All experiments were performed using cells at passages between 6 and 15.
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Characterization of Undifferentiated SGP-1. Cells seeded at a low density were positive for both ␣61 integrin and intracellular laminin staining (Fig. 2A-C). SGP-1 cells at the monolayer stage were not positive for AFP, albumin, insulin, glucagon, amylase, glial fibrillary acidic protein, and Tuj-1 (data not shown). SGP-1 cells were weakly positive for type I collagen (data not shown). Flow cytometric analysis of SGP-1 cells, shown in Fig. 3, demonstrated that the cells were positive for ␣61 integrin (Fig. 3C and D), negative for CD34, and low positive for CD44s (Fig. 3B and D). CD44s were transiently positive in epithelium of acini and ducts in the developing salivary gland, but positive cells in the adult salivary gland were not evident.31 A small number of SGP-1 cells were positive for c-kit and Thy-1 (Fig. 3B and C). These proteins are characteristic markers of hepatic oval cells.32 After confluency was achieved and cell density increased, small clusters of SGP-1 appeared. In the small clusters, the cells were highly positive for ␣61 integrin. The ratio of Thy-1–positive cells gradually increased after SGP-1 formed clusters (data not shown). SGP-1 Cells Differentiate Into Endodermal Lineage. When SGP-1 cells formed clusters containing 30 to 100 cells, they differentiated into endodermal lineage on type I collagen-coated dishes (Fig. 4) and 2 major types of clusters appeared: one consisting of hepatocyte-like cell type (hepatic cluster) and the other islet-like cell type of the pancreas (pancreatic cluster). The hepatic clusters mainly contained cells positive for AFP and/or albumin (Fig. 2D-F). The pancreatic clusters mainly contained cells positive for glucagon and/or insulin (Fig. 2G-I). In the hepatic clusters, cells with AFP usually located at the center and cells with albumin tended to present at the periphery of these clusters. The cells with albumin or AFP were rare in the pancreatic clusters. Clusters containing hepatocyte-like and islet-like cells are equally generated when cultured on type I collagencoated dishes. The ratios of the cells in the clusters positive for AFP, albumin, glucagon, and insulin were 21.6%, 19.5%, 11.1%, and 15.0%, respectively. More than 80% of the clusters had differentiated. Under these conditions of cell culture, amylase- or cytokeratin 19 –positive cells were rare (⬍5%). Cells positive for these 2 cell type markers, representing acinar and duct epithelial cells, are predominant in the adult salivary gland. On a thick Matrigel culture with hepatocyte growth factor, SGP-1 cells differentiate into cytokeratin 19 –positive cells and amylasepositive cells (data not shown). These investigations suggested that cluster formation of the cells was essential for differentiation of SGP-1 on type I collagen. When SGP-1 cells were cultured without an extracellular matrix coating, pro-
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Fig. 2. Immunofluorescent stainings of SGP-1 and endodermal lineage-committed cell containing cluster. Staining for laminin was performed (A) without Tween 20 treatment or (B) after Tween 20 treatment. (A-C) Nuclei were stained by 4⬘,6-diamidino-2-phenylindole. Insets for A and B show laminin staining without the 4⬘,6-diamidino2-phenylindole image. (C) Tween 20 –treated SGP-1 were stained (green, ␣61 integrin; red, laminin). (Inset) SGP-1 staining without Tween 20 treatment. Hepatic clusters formed by SGP-1 on (D) type I collagen or on (E and F) laminin were stained using an anti-rat albumin antibody (green; D-F) and antiAFP antibody (red; D-F). On laminincoated dishes, all cells in clusters were positive for hepatic differentiation marker, albumin, and/or AFP (E and F). Pancreatic clusters formed by SGP-1 on type I collagen were stained using an anti-insulin antibody (green; G and I) and an antiglucagon antibody (red; H and I). Scale bars: A-C, 20 m; D-I, 100 m.
liferation, cluster formation, and differentiation of the cells were impaired. When the SGP-1 cells were transferred to laminin-coated dishes at a density of 3 ⫻ 104 cells/60-mm dish and cultured for 10 days, the proliferated cells in the clusters became the hepatic cell types (Fig. 2E and F). More than 90% of SGP-1 cells were positive for albumin or AFP when cultured under these conditions. In contrast, the production of islet-like cell types was negligible. Reverse-transcription PCR analysis of messenger RNA obtained from the cultured cells on type I collagen-coated dishes showed that clusters of SGP-1 cells expressed genes for AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2.4,28,29 However, cells of monolayer sheets did not express any of these genes (Fig. 5). This observation indicates that cluster formation plays a pivotal role for SGP-1 cells to differentiate into endodermal lineage. Transplantation of SGP-1 Cells Into Liver. To test in vivo differentiation of SGP-1 cells, 1 ⫻ 106 of the cells cultured as monolayers were injected into the spleen of recipient 9-week-old female LEC rats treated according to the 2-AAF/two-thirds hepatectomy protocol as described in Materials and Methods. Donor-derived cells in recipient liver tissue were detected by in situ hybridization using sry probe. Donor-derived cells were found in hepatocytes
Fig. 3. Flow cytometric analysis of SGP-1 cells cultured on type I collagen. SGP-1 cell suspensions were incubated using the following antibodies: (A) isotype control, (B) fluorescein isothiocyanate– conjugated anti-CD34 antibody and phycoerythrin-conjugated anti– c-kit antibody, (C) anti-␣61 integrin antibody and phycoerythrin-conjugated anti–Thy-1 antibody, and (D) anti-␣61 integrin antibody and phycoerythrin-conjugated anti-CD44s antibody. 7-Aminoactinomycin D was used to discriminate living cells from dead cells, which were excluded from cytometric analysis.
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Discussion
Fig. 4. Schematic presentation for proliferation and cluster formation of SGP-1 on type I collagen-coated dish. (A) Low-density phase. SGP-1 were plated at a low density (4.0 ⫻ 105 cells/100-mm dish) on type I collagen-coated dishes. At this phase, SGP-1 were positive for intracellular laminin (Fig. 2B and C). (B) High-density phase. Six to seven days after plating, density was approximately 3.0 to 3.5 ⫻ 106 cells/100-mm dish. At this phase, clusters were not observed. (C) Formation of small cluster phase. At high density on type I collagen, SGP-1 started to form small cell clusters (Fig. 1H). There were 1.0 to 3.0 ⫻ 104 clusters formed in the 100-mm dish, and part of the cells began to differentiate. (D) Formation of large cluster phase. The SGP-1 continued to grow and formed large clusters. Differentiated cells were present only in the clusters (Fig. 2D-I). Dark cells indicate differentiated cells.
and bile duct epithelial cells 2 weeks after the transplantation (Fig. 6A and B). These cells could not be distinguished from surrounding cells. Albumin immunostaining showed that these hepatocyte-like cells were positive for albumin (Fig. 6C). Next, we labeled the SGP-1 cells with EGFP using a retrovirus vector containing EGFP complementary DNA. The labeled cells differentiated into hepatocytelike cells, islet-like cells, and neural cells in vitro as parental SGP-1 cells (data not shown). A total of 2 ⫻ 106 of SGP-1/H2 were transplanted into recipient female rats; 2 weeks later, EGFP-positive cells in the recipient liver were visible as seen under a fluorescent microscope (Fig. 6D). A portion of small lobes in the liver was surrounded by EGFP-positive cells. These cells positive for GFP were also positive for albumin immunostaining (Fig. 6E and F).
Based on transplantation studies, we identified epithelium-like cells as a possible progenitor for liver cells (SGP1). Characteristic features of undifferentiated SGP-1 are that it expresses ␣61 integrin and laminin, which are ligands for ␣61 integrin. It was shown that duct cells of the salivary gland are positive for AFP33; however, SGP-1 cells were negative for AFP. Oval cells, a tissue stem cell of the liver, are positive for AFP, albumin, and cytokeratin 19. In addition, oval cells express Thy-1, CD34, and ckit, which are markers of hematopoietic stem cells.32 In contrast, SGP-1 is negative for AFP, albumin, cytokeratin 19, and CD34. These findings indicated that SGP-1 is distinct from the hepatic oval cell. Hepatic colony-forming units in culture (H-CFU-C) is a progenitor cell for hepatic cell types obtained from fetal livers of mice.34,35 The H-CFU-C cell is negative for c-kit and CD45, positive for AFP and c-met, and low positive for CD49f.35 The property of SGP-1 differs from H-CFU-C because it is highly positive for CD49f. In addition, H-CFU-C produces cytokeratin 19 –positive cells when cultured on laminin-coated dishes; however, production of cytokeratin 19 –positive cells from SGP-1 on laminin-coated dishes or on type I collagen-coated dishes was rare. Reverse-transcription PCR analyses of messenger RNA from undifferentiated SGP-1 confirmed that the cells do not express AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2. Thus, SGP-1 does not express specific genes characteristic of differentiated hepatocytes or pan-
Fig. 5. Reverse-transcription PCR analysis of messenger RNA obtained from cultured SGP-1 cells. Total RNAs were isolated from monolayer and clusters of SGP-1. Complementary DNAs transcribed from the RNAs by reverse transcriptase were used as templates for PCR with primers for glyceraldehyde-3-phosphate dehydrogenase, AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2.
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Fig. 6. Transplantation of SGP-1 cells. Liver sections obtained from recipient rats on day 14 after cell transplantation according to the 2-AAF/two-thirds hepatectomy protocol are shown. (A-C) Transplanted SGP-1 cells were detected by in situ hybridization using sry probe. Arrows indicate sry-positive nuclei. SGP-1– derived cells were found as hepatocyte-like cells (arrows; A and C) or bile duct epithelium-like cells (arrows; B). Fifteen rats were used for these experiments, and it was shown that (A) sry-positive hepatocyte was approximately 5.7% ⫾ 3.2% and (B) sry-positive bile duct epithelium was approximately 1.5% ⫾ 2.0%. SGP-1– derived hepatocyte-like cells (arrows; C) were immunoreactive with anti-albumin antibody. (D) EGFP-labeled SGP-1 (SGP-1/H2) cells transplanted into the rat liver were detected 14 days after the transplantation using fluorescent microscopy (green; D). (E) Green fluorescent protein (GFP)-positive cells formed foci in the liver section. Five to 10 large foci were present in the liver, and many small foci had formed. Approximately 5% to 10% of the hepatocytes were GFP positive, as seen in 3 independent experiments. (F) GFP-positive cells were positive for albumin staining. Sections counterstained with (A-C) hematoxylin or (E and F) 4⬘,6-diamidino-2-phenylindole. (Original magnification: A-C, E, and F, ⫻400.)
creatic islet cells. These features are unique among previously described tissue stem cells for liver1,32,34,35 and pancreas.11,36 SGP-1 can be maintained in culture on type I collagencoated dishes for at least 3 months without losing the capacity to generate diverse cell types in culture. The cells are sensitive for irradiation-induced cell arrest and do not form a teratoma or a transformed tumor when transplanted subcutaneously into SCID mice. Single cell cultures of SGP-1 repeatedly produced original multipotent
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progenitor cells capable of differentiation into various cell types. However, once the cells are differentiated, they do not produce multipotent progenitor cells; therefore, undifferentiated cells are necessary to produce new progenitors. These results suggested that continuous renewal of multipotent SGP-1 should occur during culture on type I collagen. SGP-1 becomes hepatic cell types when transplanted via the portal vein. We performed transplantation of freshly isolated salivary gland cells or cultured SGP-1 using 2 different methods. First, we detected the donor cells by means of in situ hybridization. This procedure was used for transplantation of freshly isolated cells from the salivary gland, and we found that small numbers of the transplanted cells are present in the area of oval cell responses. Similarly, some of the SGP-1 cells are present in the oval cell response area after the transplantation. These cells could not be distinguished from the surrounding oval cell. It is possible that some of the progenitor cells in the ligated salivary glands may follow the process of differentiation of hepatic stem cells present in the liver; however, further experiments will be needed to clarify this possibility. SGP-1 is likely to be capable of differentiating into hepatic cell types when transplanted into the liver. Alternatively, the transplantation experiments using EGFP-labeled cells demonstrated that the transplanted cells show focal expansion, suggesting that SGP-1 is capable of multiplying in the liver. These results suggested that SGP-1 has the capacity of a precursor cell for hepatic cell types. The efficacy of retroviral transduction is as high as cultured fibroblasts; hence, SGP-1 may be a suitable target for gene therapy using a retrovirus vector. Differentiation of SGP-1 to hepatic cell types was achieved by culturing the cells on type I collagen-coated dishes or on laminin-coated dishes. The SGP-1 express AFP, albumin, glucagon, insulin, Pdx-1, Pax-4, Pax-6, and Nkx2.2 after the formation of clusters on type I collagen-coated dishes. Pdx-1 is one of the early genes functioning during development of the pancreas. Pax-4 and Pax-6 are homeobox genes, and expression of these genes is essential for maturation of islets. These results suggested that expression of insulin and glucagon genes are consequences of sequential activation of genes that participate in the development of islets. Further characterization of the differentiation process may clarify the precise step involved in the production of hepatic cell type form SGP-1. In this context, SGP-1 will be useful to investigate genes involved in early development of the mammalian liver. Cluster formation is essential for differentiation of SGP-1 cells in culture. In hepatic oval cells, the formation of cell clusters led to differentiation into pancreatic endo-
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crine cells when cultured in medium containing 10% fetal calf serum and 23 mmol/L glucose.4 Cell-cell interactions should play an important role in differentiation processes in stem/progenitor cells. Our experimental data indicated that the presence of extracellular matrix laminin led the SGP-1 to the hepatic cell type. Because ␣61 integrin is expressed in SGP-1, laminin should be one of the important proteins that affects the fate of SGP-1. Alternatively, on a Matrigel culture with hepatocyte growth factor, SGP-1 cells produced cytokeratin 19 –positive cells and amylase-positive cells. There is evidence to support the notion that cell fates of tissue stem cells largely depend on the extracellular matrix.37,38 SGP-1 expresses laminin. Small numbers of lamininpositive cells appeared in the ligated glands, and these cells were not seen in the nonligated glands. Laminin-positive cells are seen in the terminal clusters during development of the salivary gland.30,39,40 Another characteristic feature of SGP-1 is that the cells are low positive for CD44s. CD44s is expressed during salivary gland morphogenesis (embryonic day 16 to birth in rats), and expression of CD44s was observed in the developing pancreas. However, it is not expressed during development of the liver.31 CD44s is a receptor for hyaluronate, has a binding domain for fibronectin, glycosaminoglycans, and is involved in cell-cell interaction. These findings in the SGP-1 suggested that this cell has some immature features of salivary gland cells. Bone marrow– derived stem cells can circulate and distribute in peripheral tissues and contribute to tissue regeneration.28,41,42 It is possible that SGP-1 cells are derived from bone marrow, and we tested this possibility by bone marrow transplantation to wild-type mice using ROSA26 mice carrying the -galactosidase gene as transgenic gene donors. These experiments showed that cells proliferating in the salivary gland after ligation were not derived from donor ROSA26 mice (Hisatomi et al., manuscript in preparation). Thus, SGP-1 is likely to originate from salivary gland tissue. Clinical use of adult progenitor cells is of great interest. Isolation of tissue stem cells from accessible organs and efficient induction of differentiation into specific tissue is indispensable for clinical application for cell transplantation therapy. We showed that in vitro culture of SGP-1 on laminin successfully induced hepatocyte-like cells producing AFP and albumin. These progenitor cells from salivary glands may prove suitable for liver cell transplantation.
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