Thy1-Positive Cells Have Bipotential Ability to Differentiate into Hepatocytes and Biliary Epithelial Cells in Galactosamine-Induced Rat Liver Regeneration
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080338
ISSN1525-2191
AutoresJunko Kon, Norihisa Ichinohe, Hidekazu Ooe, Qijie Chen, Kazunori Sasaki, Toshihiro Mitaka,
Tópico(s)Pancreatic function and diabetes
ResumoIn galactosamine (GalN)-induced rat liver injury, hepatic stem/progenitor cells, small hepatocytes (SHs) and oval cells, transiently appear in the initial period of liver regeneration. To clarify the relationship between SHs and oval cells, CD44+ and Thy1+ cells were sorted from GalN-treated livers and used as candidates for SHs and oval cells, respectively. Some Thy1+ cells isolated 3 days after GalN-treatment (GalN-D3) formed CD44+ cell colonies, but those from GalN-D2 could form few. GeneChip (Affymetrix, Inc, Santa Clara, CA) analysis of the sorted cells and cultured Thy1+ cells suggested that hepatocytic differentiation progressed in the order Thy1+ (GalN-D3), Thy1+ cell colony (Thy1-C), and CD44+ (GalN-D4) cells. When Thy1+, Thy1-C, and CD44+ cells were transplanted into retrorsine/PH rat livers, they could proliferate to form hepatocytic foci. At 30 days after transplantation most cells forming the foci derived from CD44+ cells possessed C/EBPα+ nuclei, whereas only a few cells derived from Thy1-C showed this positivity. When Thy1+ (GalN-D3) cells were cultured between collagen gels in medium with hepatocyte growth factor+/dexamethasone−/dimethyl sulfoxide−, ducts/cysts consisting of biliary epithelial cells appeared, whereas with CD44+ and Thy1+ (GalN-D2) cells they did not. Taken together, these results indicate that the commitment of Thy1+ cells to differentiate into hepatocytes or biliary epithelial cells may occur between Day 2 and Day 3. Furthermore, some Thy1+ cells may differentiate into hepatocytes via CD44+ SHs. In galactosamine (GalN)-induced rat liver injury, hepatic stem/progenitor cells, small hepatocytes (SHs) and oval cells, transiently appear in the initial period of liver regeneration. To clarify the relationship between SHs and oval cells, CD44+ and Thy1+ cells were sorted from GalN-treated livers and used as candidates for SHs and oval cells, respectively. Some Thy1+ cells isolated 3 days after GalN-treatment (GalN-D3) formed CD44+ cell colonies, but those from GalN-D2 could form few. GeneChip (Affymetrix, Inc, Santa Clara, CA) analysis of the sorted cells and cultured Thy1+ cells suggested that hepatocytic differentiation progressed in the order Thy1+ (GalN-D3), Thy1+ cell colony (Thy1-C), and CD44+ (GalN-D4) cells. When Thy1+, Thy1-C, and CD44+ cells were transplanted into retrorsine/PH rat livers, they could proliferate to form hepatocytic foci. At 30 days after transplantation most cells forming the foci derived from CD44+ cells possessed C/EBPα+ nuclei, whereas only a few cells derived from Thy1-C showed this positivity. When Thy1+ (GalN-D3) cells were cultured between collagen gels in medium with hepatocyte growth factor+/dexamethasone−/dimethyl sulfoxide−, ducts/cysts consisting of biliary epithelial cells appeared, whereas with CD44+ and Thy1+ (GalN-D2) cells they did not. Taken together, these results indicate that the commitment of Thy1+ cells to differentiate into hepatocytes or biliary epithelial cells may occur between Day 2 and Day 3. Furthermore, some Thy1+ cells may differentiate into hepatocytes via CD44+ SHs. It is well known that hepatic stem/progenitor cells are activated when the proliferation of mature hepatocytes (MHs) is inhibited by hepatotoxins.1Sell S The role of progenitor cells in repair of liver injury and in liver transplantation.Wound Rep Reg. 2001; 9: 467-482Crossref PubMed Scopus (57) Google Scholar, 2Forbes S Vig P Poulsom R Thomas H Alison M Hepatic stem cells.J Pathol. 2002; 197: 510-518Crossref PubMed Scopus (143) Google Scholar, 3Fausto N Campbell JS The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (561) Google Scholar, 4Roskams TA Libbrecht L Desmet VJ Progenitor cells in diseased human liver.Semin Liver Dis. 2003; 23: 385-396Crossref PubMed Scopus (275) Google Scholar, 5Knight B Matthews VB Olynyk JK Yeoh GC Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell.BioEssays. 2005; 27: 1192-1202Crossref PubMed Scopus (35) Google Scholar, 6Walkup MH Gerber DA Hepatic stem cells: in search of.Stem Cells. 2006; 24: 1833-1840Crossref PubMed Scopus (64) Google Scholar Of these hepatic stem/progenitor cells, oval cells and small hepatocytes (SHs) are well recognized. Oval cells, named for their possession of ovoid nuclei,7Farber E Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3′-methy-4-dimethylaminoazobenzene.Cancer Res. 1956; 16: 142-149PubMed Google Scholar are known to express markers for biliary epithelial cells (BECs), eg, cytokeratin (CK) 7 and CK19, and for hepatoblasts, eg, a-fetoprotein (AFP), and cell membrane proteins such as CD34, c-kit, and Thy-1, shared hematopoietic stem cell markers.8Petersen BE Goff JP Greenberger JS Michalopoulos GK Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Crossref PubMed Scopus (336) Google Scholar Recently, the expression of delta-like protein/preadipocyte factor 1 and glypican-3 in oval cells was reported.9Jensen CH Jauho EI Santoni-Rugiu E Holmskov U Teisner B Tygstrup N Bisgaard HC Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.Am J Pathol. 2004; 164: 1347-1359Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 10Grozdanov PN Yovchev MI Dabeva MD The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells.Lab Invest. 2006; 86: 1272-1284Crossref PubMed Scopus (86) Google Scholar SHs are a subpopulation of hepatocytes.11Mitaka T Mikami M Sattler GL Pitot HC Mochizuki Y Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor.Hepatology. 1992; 16: 440-447Crossref PubMed Scopus (106) Google Scholar Their size is less than half that of MHs and they possess hepatic characteristics. These cells can clonally proliferate12Mitaka T Kojima T Mizuguchi T Mochizuki Y Growth and maturation of small hepatocytes isolated from adult liver.Biochem Biophys Res Commun. 1995; 214: 310-317Crossref PubMed Scopus (67) Google Scholar and mature by interacting with hepatic nonparenchymal cells13Mitaka T Sato F Mizuguchi T Yokono T Mochizuki Y Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (205) Google Scholar or as a result of treatment with Matrigel (BD Bioscience, Bedford, MA).14Sugimoto S Mitaka T Ikeda S Harada K Ikai I Yamaoka Y Mochizuki Y Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes.J Cell Biochem. 2002; 1: 16-28Crossref Scopus (48) Google Scholar The mature SHs express genes and proteins related to hepatic differentiated functions.14Sugimoto S Mitaka T Ikeda S Harada K Ikai I Yamaoka Y Mochizuki Y Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes.J Cell Biochem. 2002; 1: 16-28Crossref Scopus (48) Google Scholar, 15Miyamoto S Hirata K Sugimoto S Harada K Mitaka T Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes.J Gastroenterol Hepatol. 2005; 20: 865-872Crossref PubMed Scopus (13) Google Scholar, 16Oshima H Kon J Ooe H Hirata K Mitaka T Functional expression of organic anion transporters in hepatic organoids reconstructed by rat small hepatocytes.J Cell Biochem. 2008; 104: 68-81Crossref PubMed Scopus (14) Google Scholar, 17Ooe H Kon J Miyamoto S Oozone Y Ninomiya S Mitaka T Cytochrome P450 expressions of cultured rat small hepatocytes after long-term cryopreservation.Drug Metabolism and Disposition. 2006; 34: 1667-1671Crossref PubMed Scopus (13) Google Scholar Recently, we reported that CD44 and D6.1A were specifically expressed in cultured SHs and that the expression disappeared when SHs matured.18Kon J Ooe H Oshima H Kikkawa Y Mitaka T Expression of CD44 in rat hepatic progenitor cells.J Hepatol. 2006; 45: 90-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar In d-galactosamine (GalN)-induced rat liver injury, the appearance of oval cells and SHs is observed in a relatively short period, within 1 week.19Lemire JM Shiojiri N Fausto N Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine.Am J Pathol. 1991; 139: 535-552PubMed Google Scholar We observed that CD44+ SHs transiently appeared at 3 to 5 days after GalN administration and that their appearance was delayed compared with that of oval cells.18Kon J Ooe H Oshima H Kikkawa Y Mitaka T Expression of CD44 in rat hepatic progenitor cells.J Hepatol. 2006; 45: 90-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The existence of SHs has been reported in another hepatic injury model. Paku et al20Paku S Nagy P Kopper L Thorgeirsson SS 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies.Hepatology. 2004; 39: 1353-1361Crossref PubMed Scopus (106) Google Scholar used a 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH) model and demonstrated the appearance of SHs by analyzing their ultrastructure. In the retrorsine following PH (Ret/PH) model, small hepatocyte-like progenitor cells (SHPCs) have been reported to appear.21Gordon GJ Coleman WB Grisham JW Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats.Am J Pathol. 2000; 157: 771-786Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 22Best DH Coleman WB Cellular responses in experimental liver injury: possible cellular origins of regenerative stem-like cells.Hepatology. 2005; 41: 1173-1176Crossref Scopus (9) Google Scholar Furthermore, Avril et al23Avril A Pichard V Bralet M-P Ferry N Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury.J Hepatol. 2004; 41: 737-743Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar reported that after MHs were labeled with the β-galactosidase gene, β-galactosidase+ SHPCs were found in the liver lobules of the Ret/PH model rat. In contrast, by treating transgenic mice expressing hepatitis B surface antigen with retrorsine, Vig et al24Vig P Russo FP Edwards RJ Tadrous PJ Wright NA Thomas HC Alison MR Forbes SJ The source of parenchymal regeneration after chronic hepatocellular liver injury in mice.Hepatology. 2006; 43: 316-324Crossref PubMed Scopus (108) Google Scholar demonstrated that oval cells could change into SHPCs. Therefore, the differentiation of oval cells into SHs is still controversial. In the present study we used the GalN-injury model to clarify whether oval cells could differentiate into hepatocytes or BECs. To resolve this issue, we used Thy1+ and CD44+ cells isolated from GalN-treated rat livers as oval cells and SHs, respectively. Many CD44+ cells isolated from 4 days after GalN-treatment (GalN-D4) could form CD44+ SH colonies, whereas some Thy1+ (GalN-D3) cells could form CD44+ SH colonies but those from GalN-D2 could form few. When isolated cells were transplanted into Ret/PH rat livers, both CD44+ and Thy1+ cells could proliferate to form foci. However, the size of foci was much larger for CD44+ cells than for Thy1+ cells. GeneChip (Affymetrix, Inc, Santa Clara, CA) analysis of gene expression suggested that hepatic differentiation progressed in the order Thy1+ (GalN-D3), cultured Thy1+ cells (Thy1-C), and CD44+ (GalN-D4) cells. In addition, CD44+ cells, SHs, and Thy1-C may possess very similar characteristics, but they are clearly different from MHs. When the sorted cells were cultured between collagen gels in medium with hepatocyte growth factor (HGF)+/dexamethasone (Dex)−, some Thy1+ cells could form ducts/cysts consisting of BECs and differentiate hepatocytes. In the present experiment, the commitment of Thy1+ cells to hepatocytic or biliary differentiation occurred between Day 2 and Day 3 after GalN-treatment. Some Thy1+ cells may differentiate into hepatocytes via CD44+ SHs. Male F344 rats (dipeptidylpeptidase IV [DPPIV]+ strain; Sankyo Lab Service Corporation, Inc, Tokyo, Japan) weighing 150 to 200 g were used. All animals received humane care and the experimental protocol was approved by the Committee of Laboratory Animals according to Sapporo Medical University guidelines. For GalN-injured livers, GalN (Acros, Geel, Belgium; 75 mg/100 g body weight dissolved in PBS) was intraperitoneally administered.18Kon J Ooe H Oshima H Kikkawa Y Mitaka T Expression of CD44 in rat hepatic progenitor cells.J Hepatol. 2006; 45: 90-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar For the transplantation experiment, female F344 rats (DPPIV− strain; Charles River, Wilmington, MA) were intraperitoneally given two injections of retrorsine (30 mg/kg body weight; Sigma Chemical Co, St. Louis, MO), 2 weeks apart.21Gordon GJ Coleman WB Grisham JW Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats.Am J Pathol. 2000; 157: 771-786Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Four weeks after the second injection, two-thirds PH was performed. Sorted DPPIV+ cells (5 × 105 cells) and colonies (about 1 × 104 colonies) from cultured Thy1+ cells were transplanted into Ret/PH livers (DPPIV−) via the spleen (three to five rats per group). Rats were used to isolate hepatic cells by the collagenase perfusion method as previously described.25Chen Q Kon J Ooe H Sasaki K Mitaka T Selective proliferation of rat hepatocyte progenitor cells in serum-free culture.Nat Protoc. 2007; 2: 1197-1205Crossref PubMed Scopus (45) Google Scholar After the perfusion, the cell suspension was centrifuged at 50 × g for 1 minute. The supernatant and the precipitate were used for sorting Thy1+ and CD44+ cells and preparing MHs, respectively.18Kon J Ooe H Oshima H Kikkawa Y Mitaka T Expression of CD44 in rat hepatic progenitor cells.J Hepatol. 2006; 45: 90-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The supernatant was centrifuged at 50 × g for 1 minute again. After this procedure was repeated, the supernatant was centrifuged at 50 × g for 5 minutes. The precipitate was suspended in PBS containing 2 mmol/L EDTA and 0.5% bovine serum albumin. Antibodies used for cell sorting are listed in Table 1. First, 2 μg/ml of anti-Thy1 or 625 ng/ml of anti-CD44 antibodies was added to the cell suspension and then a microbead-labeled secondary antibody was added. The suspension volume and incubation time were followed by manufacturer's information. Magnetic separation was done by using a MidiMACS separation unit (Miltenyi Biotec, Bergisch Gladbach, Germany). After the number of viable cells was counted, 1 × 105 viable cells were plated in a 12-well plate (Corning, Corning, NY) and cultured in the medium listed in Table 2. Some sorted Thy1+ cells were cultured for 5 days and epithelial cell colonies were collected as previously reported.17Ooe H Kon J Miyamoto S Oozone Y Ninomiya S Mitaka T Cytochrome P450 expressions of cultured rat small hepatocytes after long-term cryopreservation.Drug Metabolism and Disposition. 2006; 34: 1667-1671Crossref PubMed Scopus (13) Google Scholar The isolated colonies were used for the GeneChip (Affymetrix, Inc) analysis and transplantation. The selective isolation of the colonies is shown in Supplement Figure S1, see http://ajp.amjpathol.org.Table 1List of Antibodies Used in the Present ExperimentsAntibodiesCompany or producerDilutionMouse anti-rat CD44BD Biosciences PharMingen, Franklin Lakes, NJ1:1000Rabbit anti-rat C/EBPαSanta Cruz, Santa Cruz, CA1:400Rabbit anti-rat CK19Generous gift from Professor Atsushi Miyajima*From the Institute of Molecular and Cellular Biology, University of Tokyo, Tokyo, Japan.1:1000Mouse anti-rat Thy1.1Serotec, Raleigh, NC1:500Goat anti-rat desminSanta Cruz1:200Mouse anti-rat SE1Immuno-Biological Lab, Takasaki, Japan1:200Rabbit anti-human, mouse, rat CD3Abcam, Tokyo, Japan1:500Rat anti-mouse IgG1 microbeadsMiltenyi Biotec, Bergisch Gladbach, Germany1:50Rat anti-mouse IgG2a+bmicrobeadsMiltenyi Biotec1:50Biotinylated anti-mouse IgG (H+L)Vector Laboratories, Burlingame, CA1:200Alexa488-conjugatedMolecular Probes, Eugene, OR1:500Alexa594-conjugatedMolecular Probes1:500* From the Institute of Molecular and Cellular Biology, University of Tokyo, Tokyo, Japan. Open table in a new tab Table 2Ingredients in Culture MediumIngredients with final concentration added to modified DMEM*Modified DMEM included 20 mmol/L HEPES, 30 mg/L proline, 25 mmol/L NaHCO3, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, 0.5 mg/L insulin, and antibiotics (penicillin, streptomycin, and gentamicin).For dish cultureFor gel culture (control)For gel culture (Dex−/HGF+)10 ng/ml HGF−−+10−7 mol/L Dex++−1% DMSO (4 days after plating)−+−* Modified DMEM included 20 mmol/L HEPES, 30 mg/L proline, 25 mmol/L NaHCO3, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, 0.5 mg/L insulin, and antibiotics (penicillin, streptomycin, and gentamicin). Open table in a new tab For collagen-sandwich culture, 8.5 ml of rat tail collagen (500 μg of dried tendon/ml 0.1% acetic acid) was mixed with 2 ml of NaOH/Dulbecco's modified Eagle's medium (DMEM) solution (0.34 N NaOH:10×DMEM = 10:3) and 500 μl aliquots of collagen/NaOH/DMEM solution were poured into 12-well plates. To gelatinize, plates were placed at 37°C for 30 minutes. After 1 × 106 cells were plated and incubated in a CO2-incubator for 1 hour, 300 μl aliquots of the collagen/NaOH/DMEM solution were added. These cells were cultured in the control and the Dex−/HGF+ medium listed in Table 2. The medium was replaced with fresh medium three times a week. Differences of the expression profiles of cells were analyzed by using an oligo microarray spotted with 31,000 probes (GeneChip Rat Genome 230 2.0 Array; Affymetrix, Inc). Total RNAs were prepared by using a GenElute-mRNA midiprep kit (Sigma). Analysis of the array data were performed with MultiExperiment Viewer (TM4, http://www.tm4.org/) by Daiichi Pure Chemical Co, Ltd (Tokai, Ibaraki, Japan). For detecting CD44+ colonies, immunocytochemistry was performed at day 10. Cells were fixed with cold absolute ethanol. After blocking the intrinsic peroxidase with 0.2% H2O2/methanol and then nonspecific staining with BlockAce (Dainippon Pharmaceuticals Co, Osaka, Japan), cells in the dish were incubated with primary antibodies at room temperature for 1 hour, followed by the avidin-biotin peroxidase complex method (Vectastain ABC Elite Kit; Vector Laboratories, Inc, Burlingame, CA). 3′-Diaminobenzidine (Tokyo Kasei Industries, Tokyo, Japan) was used as a substrate. Nuclei of the cells were counterstained with hematoxylin. The number of CD44+ colonies at day 10 was counted and the positivity was calculated. Three separate experiments were performed. For fluorescent immunocytochemistry, livers and sandwich-cultured cells were frozen by using isopentane/liquid nitrogen and the samples were kept at −80°C until use. Seven-micrometer-thick sections were prepared and dried. To determine the characteristics of sorted cells, isolated cells were cultured on CultureSlides (Becton Dickinson Labware, Franklin Lakes, NJ) for 2 days. After blocking, the sections were incubated with primary antibodies for 1 hour at room temperature and then fluorescent-conjugated secondary antibodies were applied for 30 minutes. All antibodies used for immunostaining were listed in Table 1. The sections were embedded with 90% glycerol including 0.01% p-phenylenediamine 4,6-diamidino-2 -phenylindole (DAPI). A confocal laser microscope (Olympus, Tokyo, Japan) was used for observation and findings were analyzed by using DP Manager (Olympus). The number of the positive cells at day 2 was counted and the positivity was calculated. More than 200 cells per dish were counted and three separate experiments were performed. To identify the donor cells, enzyme histochemistry for DPPIV was performed. DPPIV enzyme activity was detected as previously described.26Shibata C Mizugichi T Kikkawa Y Nobuoka T Oshima H Kawasaki H Kawamoto M Katsuramaki T Mitaka T Hirata K Liver repopulation and long-term function of rat small hepatocyte transplantation as an alternative cell source for hepatocyte transplantation.Liver Transpl. 2006; 12: 78-87Crossref PubMed Scopus (26) Google Scholar Briefly, sections were fixed in cold 10% buffered formalin for 5 minutes and then cold ethanol for 5 minutes and air-dried. The samples were incubated for 60 minutes in a substrate solution containing 0.5 mg/ml gly-pro-methoxy-β-naphthylamide (Sigma), 1.0 mg/ml Fast Blue BB (Sigma), and 0.1M PBS (pH 6.5). All samples were counterstained with hematoxylin and mounted in glycerol. DPPIV+ foci were photographed by using a microscope equipped with a CCD camera and the area of each focus was measured by using Image J (http://rsb.info.nih.gov/ij/index.html). After DPPIV staining, some samples were immunohistochemically stained. Primary antibodies for CD44, SE1, CK19, and CCAAT/Enhancer Binding Protein (C/EBP) α were used for clarifying the characteristics of DPPIV+ foci formed by donor cells. To detect the primary antibody, avidin-biotin complex method or fluorescent immunohistochemistry were performed as described above. The cells in the collagen gel were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH7.4) at room temperature for 30 minutes, postfixed in 2% osmium tetroxide in the buffer, dehydrated by graded ethanol, and embedded in situ in Epon812. The procedure was previously described in detail.13Mitaka T Sato F Mizuguchi T Yokono T Mochizuki Y Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (205) Google Scholar Statistical analysis was performed by using Student's t-test. A P value of <0.05 was considered significant. To characterize the sorted cells, we performed two experiments. The sorted cells from GalN-D3 were cultured for 2 days and immunocytochemistry for albumin, CD44, CK19, Thy1, and desmin was conducted. As shown in Figure 1A, the morphology of Thy1+ cells was roughly divided into two types, polygonal (epithelial-like) and spindle (fibroblast-like) shaped. Most of the spindle shaped cells were desmin+ and the percentage of Thy1+/desmin+ cells was about 44.2 (Table 3). On the other hand, the polygonal cells were albumin, CD44, or CK19+. The percentages of Thy1+/CK19+, Thy1+/albumin+, and Thy1+/CD44+ cells were 16.2, 30.0, and 38.3, respectively. In addition, the epithelial-like cells had a tendency to form aggregates consisting of a small number of cells at the time of sorting. The epithelial-like cells rapidly proliferated and formed a colony (Figure 1B). The morphology of cells in the colonies was divided into two types, cells with compact and granular cytoplasm, and others that were flat with few granules in their cytoplasm. The former were CD44+/desmin− and the latter CD44−/desmin+. On the other hand, most sorted CD44+ cells were polygonal and their morphology had epithelial-like features. Some cells, about 15%, were fibroblastic and positive for desmin (Figure 1C). The percentages of CD44+/CK19+, CD44+/albumin+, and CD44+/Thy1+ cells were 60.1, 62.5, and 65.0, respectively. CD44+/albumin+ cells (Figure 1C) were larger than the Thy1+/albumin+ ones (Figure 1A), whereas the size and features of CD44+/CK19+ cells (Figure 1C) were similar to those of Thy1+/CK19+ ones (Figure 1A).Table 3Characterization of Sorted CellsCellsCombinationPopulation, %Sorted Thy1+ cells Thy1+71.6 ± 12.2Thy1+ / CK19+16.2 ± 4.7Thy1+ / Desmin+44.2 ± 25.1Thy1+ / Albumin+30.0 ± 12.0Thy1+ / CD44+38.3 ± 17.1 Thy1−Thy1− / CK19+3.6 ± 3.2Thy1− / Desmin+1.1 ± 1.3Thy1− / Albumin+3.9 ± 6.7Thy1− / CD44+0Sorted CD44+ cells CD44+81.9 ± 5.8CD44+ / CK19+60.1 ± 14.1CD44+ / Desmin+14.9 ± 2.5CD44+ / Albumin+62.5 ± 6.1CD44+/ Thy1+65.0 ± 4.4 CD44−CD44− / CK19+5.4 ± 1.2CD44− / Desmin+2.3 ± 3.3CD44− / Albumin+6.0 ± 8.6CD44− / Thy1+22.1 ± 0.9 Open table in a new tab Sorted Thy1+ and CD44+ cells were cultured for 10 days and the formation of SH colonies was examined by CD44 immunostaining (Figure 2, A and B). CD44+ colonies were regarded as SH colonies. Although sorted Thy1+ (GalN-D2) cells were cultured for 10 days, they could rarely form CD44+ colonies, some Thy1+ (GalN-D3) cells formed morphologically typical SH colonies, all cells of which were positive for CD44 (Figure 2A). The frequency of the formation and the average area of CD44+ colonies were calculated and the data are shown in Table 4. Thy1+ (GalN-D3) cells could form SH colonies more efficiently than those from GalN-D2 and GalN-D4. Although the efficiency of the colony formation was two times greater for Thy1+ (GalN-D3) cells than for those from GalN-D4, that of Thy1+ (GalN-D3) cells had half the efficiency of CD44+ (GalN-D3) cells. Thy1+ (GalN-D4) cells could form about twofold larger colonies than those from GalN-D3. The average sizes of the colonies formed by Thy1+ (GalN-D4) cells and CD44+ (GalN-D3) cells were about the same but only about half as large as those formed by CD44+ (GalN-D4) cells. These results showed that, although most Thy1+ (GalN-D2 cells) were not committed to a hepatic lineage, some Thy1+ (GalN-D3, 4) cells may have already differentiated into CD44+ SHs.Table 4Efficiency of SH Colony FormationCellsDays after GalNAttached cells at D1 (×104 cells)CD44+ colonies at D10 (colonies)Efficiency*Efficiency = (Number of colonies/Attached cells)/104.Average area of CD44+ colonies at D10 (× 10−3 mm2)Thy121.6 ± 0.21 ± 0.80.6—32.0 ± 0.494.7 ± 10.347.4263.0 ± 91.541.0 ± 0.223.0 ± 3.623.9448.8 ± 401.9†P < 0.05 when compared to Thy1+ (GalN-D3) cells.CD4421.8 ± 0.20.7 ± 0.50.4—31.7 ± 0.2163.0 ± 24.795.9520.0 ± 477.940.9 ± 0.1184.0 ± 12.1195.61063.6 ± 331.8‡P < 0.05 when compared to CD44+ (GalN-D3) cells.§P < 0.05 when compared to Thy1+ (GalN-D4) cells.Three independent experiments were carried out and three wells per experiments were counted.* Efficiency = (Number of colonies/Attached cells)/104.† P < 0.05 when compared to Thy1+ (GalN-D3) cells.‡ P < 0.05 when compared to CD44+ (GalN-D3) cells.§ P < 0.05 when compared to Thy1+ (GalN-D4) cells. Open table in a new tab Three independent experiments were carried out and three wells per experiments were counted. To investigate whether the sorted cells could differentiate into MHs, they were transplanted into Ret/PH-rat livers. As shown in Figure 3, A–C, DPPIV+ cells were found in recipient livers of all groups. To characterize the cells forming DPPIV+ foci, double-staining was performed. As shown in Figure 4A, CK19+ cells were observed in the portal area and some cells comprised small bile ductules. Although most DPPIV+ cells did not show CK19-positivity, some CK19+ small ductules were located in the edge of the DPPIV+ cluster. SE1+ cells were observed in sinusoids, not in DPPIV+ cells (Figure 4B). Although DPPIV+ cells did not show SE1-positivity, the number of SE1+ cells within the foci was fewer than that around the foci (Figure 4, N–P). The expression of CD44 was lost in the DPPIV+ cells at 30 days after transplantation (Figure 4C). Only BECs in ductules expressed CD44. CD3+ T cells and CK19+ BECs were not observed within DPPIV+ foci (Figure 4, H–M), while most DPPIV+ cells possessed C/EBPα+ nuclei (Figure 4, D–G). The number and the area of DPPIV+ foci were measured and the data are summarized in Table 5. Although there was no significant difference in number between Thy1+ (GalN-D2) and (GalN-D3) cells, the average area of the foci formed by Thy1+ (GalN-D3) cells was about twice that for GalN-D2. The average number and area derived from CD44+ (GalN-D4) cells were about 10-fold and twofold greater than those derived from Thy1+ (GalN-D3) cells, respectively.Figure 4Characteristics of DPPIV+ foci in the livers after CD44+ cell transplantation. Frozen sections were obtained from livers at 30 days after transplantation of sorted CD44+ cells. Immunohistochemistry and DPPIV-enzyme histochemistry are shown: A combination of DPPIV-enzyme histochemistry and immunohistochemistry for CK19 (A); SE1 (B); CD44 (C); and C/EBPα (D). E–M: Double-fluorescent immunohistochemistry for DPPIV (E, H, K) and C/EBPα (F), CD3 (I), or CK19 (L). N–P: A combination of DPPIV-enzymatic histochemistry (N) and fluorescent immunohistochemistry for SE1 (O). Merged images are combined with DAPI-staining (G, J, M, P). Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 5Number and Area of Transplanted DPPIV+ FociDays after GalNNCellsAverage number of DPPIV+ foci per whole liver area (× 10−3 mm2)Average of DPPIV+ area (× 10−3 mm2)25Thy113.5 ± 0.250.4 ± 20.235Thy111.4 ± 0.2118.9 ± 54.245CD44118.1 ± 0.3*P < 0.05 when compared to either D2 or D3.209.5 ± 131.6* P < 0.05 when compared to either D2 or D3. Open table in a new tab When Thy1+ (GalN-D3) cell-derived colonies cultured for 5 days (Thy1-C) were transplanted into Ret/PH-
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