Gastric Mesenchymal Myofibroblasts Maintain Stem Cell Activity and Proliferation of Murine Gastric Epithelium in Vitro
2014; Elsevier BV; Volume: 185; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2014.11.007
ISSN1525-2191
AutoresTakahito Katano, Akifumi Ootani, Tsutomu Mizoshita, Satoshi Tanida, Hironobu Tsukamoto, Keiji Ozeki, Hiromi Kataoka, Takashi Joh,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoStem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymal myofibroblast (GMF) cell line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen gel. Cultured stomach cells yielded expanding sphere-like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures (P = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5+ stem cells in the cultured gastrospheres. Lgr5+ cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GMFs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo. Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs (P = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts (P = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5+ stem cell activity and affecting normal gastric epithelial differentiation and proliferation. Stem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymal myofibroblast (GMF) cell line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen gel. Cultured stomach cells yielded expanding sphere-like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures (P = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5+ stem cells in the cultured gastrospheres. Lgr5+ cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GMFs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo. Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs (P = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts (P = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5+ stem cell activity and affecting normal gastric epithelial differentiation and proliferation. The homeostasis of the gastrointestinal mucosa is maintained by a variety of mechanisms. Epithelial cells of the gastric mucosa are organized in vertical flask-shaped structures called glands, which are composed of several epithelial cell types, including surface mucous (foveolar) cells, mucous neck cells, parietal cells, zymogenic (chief) cells, and enteroendocrine cells.1Karam S.M. Leblond C.P. Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell.Anat Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar, 2Karam S.M. Leblond C.P. Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells.Anat Rec. 1993; 236: 280-296Crossref PubMed Scopus (167) Google Scholar The vertical unit of the gastric mucosa is composed of the pit, isthmus, neck, and base.1Karam S.M. Leblond C.P. Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell.Anat Rec. 1993; 236: 259-279Crossref PubMed Scopus (323) Google Scholar, 2Karam S.M. Leblond C.P. Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells.Anat Rec. 1993; 236: 280-296Crossref PubMed Scopus (167) Google Scholar The glands are surrounded by supporting stromal cells. 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Yasugi S. BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression.Development. 2000; 127: 981-988PubMed Google Scholar, 14Fukamachi H. Kato S. Asashima M. Ichinose M. Yuasa Y. Activin A regulates growth of gastro-intestinal epithelial cells by mediating epithelial-mesenchymal interaction.Dev Growth Differ. 2013; 55: 786-791Crossref Scopus (7) Google Scholar In vitro analysis of gastric epithelial growth and differentiation has been hampered by a lack of appropriate culture systems that recapitulate the in vivo environment. Several researchers have put a great deal of effort into generating long-term cultures of primary gastric epithelium, but the maintenance of gastric epithelial viability for more than 14 days is difficult, and the three-dimensional (3D) architecture of the gastric glands is not preserved.15Terano A. Ivey K.J. Stachura J. Sekhon S. Hosojima H. McKenzie Jr., W.N. Krause W.J. Wyche J.H. Cell culture of rat gastric fundic mucosa.Gastroenterology. 1982; 83: 1280-1291Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 16Sanders M.J. Amirian D.A. Ayalon A. Soll A.H. Regulation of pepsinogen release from canine chief cells in primary monolayer culture.Am J Physiol. 1983; 245: G641-G646PubMed Google Scholar, 17Rattner D.W. Ito S. Rutten M.J. Silen W. A rapid method for culturing guinea pig gastric mucous cell monolayers.In Vitro Cell Dev Biol. 1985; 21: 453-462Crossref PubMed Scopus (35) Google Scholar, 18Chew C.S. Parietal cell culture: new models and directions.Annu Rev Physiol. 1994; 56: 445-461Crossref PubMed Scopus (44) Google Scholar, 19Ootani A. Toda S. Fujimoto K. Sugihara H. Foveolar differentiation of mouse gastric mucosa in vitro.Am J Pathol. 2003; 162: 1905-1912Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Therefore, current studies of gastric stem cells and their niche largely rely on the use of cell lines or studies in genetically manipulated mice, which are expensive and time consuming.20Barker N. Huch M. Kujala P. van de Wetering M. Snippert H.J. van Es J.H. Sato T. Stange D.E. Begthel H. van den Born M. Danenberg E. van den Brink S. Korving J. Abo A. Peters P.J. Wright N. Poulsom R. Clevers H. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Cell Stem Cell. 2010; 6: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar Here, we describe a novel long-term method for culturing primary mouse glandular stomach cells to observe the dynamic effects of mesenchymal myofibroblasts in the stem cell niche on the differentiation of gastric epithelium. By using our culture method, we examined the effect of gastric mesenchymal myofibroblasts (GMFs) on the growth of gastric epithelial cells and found that GMFs contribute to the long-term maintenance of stem cell activity and proliferation of the gastric epithelium in vitro. Then, we identified tissue-specific factors in GMFs and intestinal mesenchymal myofibroblasts (IMFs). This method will enable the study of gastric epithelial-mesenchymal interactions, as well as investigation of normal gastric epithelial growth and differentiation. The experimental design of this study was approved by the Animal Care Committee of the Nagoya City University Animal Research Institute. The animals were cared for in accordance with institutional guidelines, which complied with the instructions of the Health, Labor and Welfare Ministry concerning animal experiments. We used the 3D culture system as previously reported.21Ootani A. Li X. Sangiorgi E. Ho Q.T. Ueno H. Toda S. Sugihara H. Fujimoto K. Weissman I.L. Capecchi M.R. Kuo C.J. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat Med. 2009; 15: 701-706Crossref PubMed Scopus (615) Google Scholar, 22Katano T. Ootani A. Mizoshita T. Tanida S. Tsukamoto H. Ozeki K. Ebi M. Mori Y. Kataoka H. Kamiya T. Toda S. Joh T. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche.Biochem Biophys Res Commun. 2013; 432: 558-563Crossref PubMed Scopus (37) Google Scholar This system maintains cultured cells that are embedded in a collagen gel in an air-liquid interface environment. Cellmatrix type I-A (Nitta Gelatin, Inc., Osaka, Japan), Ham's F-12, and sterile reconstitution buffer (2.2 g NaHCO3 in 100 mL of 0.05 N NaOH and 200 mmol/L HEPES) were mixed at a ratio of 8:1:1. This reconstituted collagen solution (1.2 mL) was poured into an inner 30-mm diameter dish (Millicell Culture Plate Inserts, PICM03050; Millicell-CM, Millipore, Billerica, MA). After 30 minutes at 37°C, the collagen gel in the inner dish had solidified, and the tissue was prepared. The glandular stomach was removed from postnatal day 2 C57BL/6J mice, placed on ice, and immediately washed in phosphate-buffered saline (PBS). Within 5 minutes, the washed tissue was minced into pieces smaller than 0.3 mm3 and mixed with the reconstituted collagen solution as described earlier. The tissue from one neonatal mouse was mixed in one dish. All work beginning from tissue removal until this step was performed on ice. The cell-containing collagen gel (1.2 mL) was poured onto the collagen gel in the inner dish, which was placed in a 60-mm diameter outer dish. After the cell-containing gel solidified, 2.0 mL Ham's F12 supplemented with 20% fetal bovine serum and 50 μg/mL gentamicin was poured into the outer dish, which was covered and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Culture medium was replaced every 5 days. This culture system, which contained only mouse stomach tissue, was considered the control culture. The glandular stomach and the middle part of the small intestine were removed from postnatal day 2 C57BL/6J mice, placed on ice, and immediately immersed and washed in PBS. The washed tissue was minced into pieces smaller than 0.5 mm3 in the dishes (Becton Dickinson Bioscience, Franklin Lakes, NJ) and incubated in culture medium (Dulbecco's modified Eagle's medium and Ham's F12 in a 1:1 ratio, supplemented with 20% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B) at 37°C in a humidified atmosphere of 5% CO2 in air. Minced pieces were grown attached to a dish. After 3 to 7 days, spindle-shaped myofibroblasts had migrated from the minced tissue fragments and grown into confluent monolayers. These cells were passaged for subculturing and used as a source of GMFs and IMFs. For the co-culture system, 5 × 105 GMFs were included under the collagen gel layer (1.2 mL) in the 3-cm inner dish. After the gel containing the GMFs solidified, the minced glandular stomach from one postnatal day 2 C57BL/6J mouse and another aliquot of 5 × 105 GMFs were mixed in 1.2 mL reconstituted collagen solution. The collagen gel containing minced tissue and the GMFs (1.2 mL) was poured onto the GMF-only collagen gel in the inner dish. Reconstituted collagen solution, culture medium, and culture conditions were the same as the control culture described earlier. Cultured cells in the collagen gel were fixed in 10% formalin, processed routinely, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. To detect mucous substances, sections were stained with periodic acid-Schiff. Immunohistochemical and immunofluorescent staining was performed with antibodies against the following antigens: MUC5AC, MUC6, human gastric mucin (HGM), HIK1083, CD10 (1:100 each; Novocastra Laboratories, Newcastle upon Tyne, UK), MUC2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), chromogranin A (1:1000; Yanaihara Institute, Inc., Fujinomiya, Japan), anti–proton pump/H+, K+-adenosine triphosphatase (ATPase) (ready to use; MBL Co., Ltd., Nagoya, Japan), pepsinogen (1:10,000; Abcam, Tokyo, Japan), proliferating cell nuclear antigen (1:100; Dako, Glostrup, Denmark), Ki-67 (1:2000; Novocastra Laboratories), α-smooth muscle actin (α-SMA) (1:200; Epitomics, Burlingame, CA), vimentin (1:250; Abcam), and cytokeratin (ready to use; Nichirei, Tokyo, Japan). The immunostaining procedures for paraffin sections were performed as described.23Mizoshita T. Tsukamoto T. Nakanishi H. Inada K. Ogasawara N. Joh T. Itoh M. Yamamura Y. Tatematsu M. Expression of Cdx2 and the phenotype of advanced gastric cancers: relationship with prognosis.J Cancer Res Clin Oncol. 2003; 129: 727-734Crossref PubMed Scopus (125) Google Scholar, 24Itoh K. Kataoka H. Sasaki M. Tanida S. Oshima T. Ogasawara N. Ohara H. Nakao H. Joh T. Bone morphogenetic protein 2 induced differentiation toward superficial epithelial cells in the gastric mucosa.J Gastroenterol. 2006; 41: 1064-1075Crossref PubMed Scopus (7) Google Scholar For immunofluorescence, GMF cells grown on culture slides (Becton Dickinson Bioscience) were washed twice with PBS, fixed for 15 minutes in fresh 3% paraformaldehyde diluted in PBS, permeabilized for 5 minutes in 0.5% Triton X-100 (Wako Pure Chemical Industries, Osaka, Japan) in PBS, blocked for 60 minutes at room temperature in 10% normal goat serum, and then incubated in primary antibody for 60 minutes at room temperature. The slides were incubated with the corresponding secondary antibodies for 60 minutes at room temperature.23Mizoshita T. Tsukamoto T. Nakanishi H. Inada K. Ogasawara N. Joh T. Itoh M. Yamamura Y. Tatematsu M. Expression of Cdx2 and the phenotype of advanced gastric cancers: relationship with prognosis.J Cancer Res Clin Oncol. 2003; 129: 727-734Crossref PubMed Scopus (125) Google Scholar, 24Itoh K. Kataoka H. Sasaki M. Tanida S. Oshima T. Ogasawara N. Ohara H. Nakao H. Joh T. Bone morphogenetic protein 2 induced differentiation toward superficial epithelial cells in the gastric mucosa.J Gastroenterol. 2006; 41: 1064-1075Crossref PubMed Scopus (7) Google Scholar The slides then were washed again and mounted with fluorescence mounting medium (Dako). As a positive control, normal mouse stomach and intestine were used for immunohistochemistry and immunofluorescence. The controls always showed positive results in a cell type–specific manner. Stained cells were observed with a confocal laser microscope (Nikon A1 Confocal System; Nikon Instech Co., Ltd., Tokyo, Japan), and data were analyzed using NIS element imaging software version 4.13 (Nikon Instech Co., Ltd.). Band-pass emission filters of 505 to 530 nm and 650 nm were used. In situ hybridization for Lgr5 was performed using the RNAscope FFPE assay kit (Advanced Cell Diagnostics, Inc., Hayward, CA) according to the manufacturer's instructions. Briefly, 4- to 5-μm–thick formalin-fixed, paraffin-embedded tissue sections were pretreated with heat and protease digestion and then hybridized with a target probe for Lgr5. Horseradish-peroxidase–based amplification steps were performed. To detect target RNA, 3,3′-diaminobenzidine tetrahydrochloride was added. Positive staining was detected as brown punctate dots. The housekeeping gene peptidylprolyl isomerase B was used as a positive control. The DapB gene was used as a negative control. Muc5ac, Muc6, Pga5, PgC, Atp4a, Chga, and glyceraldehyde-3-phosphate dehydrogenase mRNA expression in gastrospheres cultured in the control system and the co-culture system was measured with real-time RT-PCR. In GMFs and IMFs, we compared mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase and 46 target genes related to morphogenesis, the hedgehog signaling pathway, bone morphogenetic protein, fibroblast growth factor, Notch, homeobox genes, and Wnt signaling (Table 1). Glyceraldehyde-3-phosphate dehydrogenase was chosen as an endogenous control to normalize the expression data. mRNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Tokyo, Japan) according to the manufacturer's instructions. TaqMan Gene Expression Assays for target genes and glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) were purchased from Applied Biosystems, and real-time quantitative RT-PCR analyses were performed in triplicate using an ABI 7500 Fast Real-Time PCR system (Applied Biosystems) according to the manufacturer's recommendations. All data are presented as fold changes of the internal control.Table 1Forty-Six Target Genes Used for Real-Time RT-PCR of Cultured MyofibroblastsGene accessionGene symbolGene descriptionMm01353100_m1Barx1BarH-like homeobox 1Mm00432087_m1Bmp4Bone morphogenetic protein 4Mm00432102_m1Bmp7Bone morphogenetic protein 7Mm00552900_m1BocBiregional cell adhesion molecule–related/down-regulated by oncogenes (Cdon) binding proteinMm00477680_m1Btrcβ-Transducin repeat–containing proteinMm00491185_m1CdonCell adhesion molecule–related/down-regulated by oncogenesMm00483039_m1Ctnnb1Catenin (cadherin-associated protein), β 1Mm01310203_m1DhhDesert hedgehogMm00433275_m1Fgf10Fibroblast growth factor 10Mm00438917_m1Fgf4Fibroblast growth factor 4Mm00546194_s1Foxc2Forkhead box C2Mm00515793_m1Foxf2Forkhead box F2Mm00433409_s1Fzd7Frizzled homolog 7 (Drosophila)Mm01700206_g1Gas1Growth arrest specific 1Mm00494654_m1Gli1GLI-Kruppel family member GLI1Mm01293111_m1Gli2GLI-Kruppel family member GLI2Mm00492345_m1Gli3GLI-Kruppel family member GLI3Mm00469580_m1HhipHedgehog-interacting proteinMm04213381_s1Hoxa5Homeobox A5Mm00439369_m1Hoxc8Homeobox C8Mm00439613_m1IhhIndian hedgehogMm00496902_m1Jag1Jagged 1Mm00550265_m1Lef1Lymphoid-enhancer binding factor 1Mm00442942_m1LifrLeukemia inhibitory factor receptorMm00464608_m1Lrp1Low-density lipoprotein receptor–related protein 1Mm02384862_g1NanogNanog homeoboxMm00476081_m1Nkx3-2NK3 homeobox 2Mm00435249_m1Notch1Notch 1Mm00436026_m1Ptch1Patched homolog 1Mm00436047_m1Ptch2Patched homolog 2Mm00489161_m1Sfrp1Secreted frizzled-related protein 1Mm01213947_m1Sfrp2Secreted frizzled-related protein 2Mm00436528_m1ShhSonic hedgehogMm00484723_m1Smad1SMAD family member 1Mm00487530_m1Smad2SMAD family member 2Mm01162710_m1SmoSmoothened homolog (Drosophila)Mm01300162_m1Sox10SRY-box containing gene 10Mm00448840_m1Sox9SRY-box containing gene 9Mm01219775_m1Stat3Signal transducer and activator of transcription 3Mm00489385_m1SufuSuppressor of fused homolog (Drosophila)Mm00437328_m1Wnt11Wingless-related MMTV integration site 11Mm00437337_m1Wnt3aWingless-related MMTV integration site 3AMm00437347_m1Wnt5aWingless-related MMTV integration site 5AMm00460518_m1Wnt9aWingless-type MMTV integration site 9AMm00656094_m1Zic1Zinc finger protein of the cerebellum 1Mm01226725_g1Zic2Zinc finger protein of the cerebellum 2MMTV, mouse mammary tumor virus. Open table in a new tab MMTV, mouse mammary tumor virus. To calculate the percentage of Ki-67+ cells, 500 cultured epithelial cells were counted, and the percentage of Ki-67+ nuclei was calculated. The data obtained from five to six independent experiments were analyzed with analysis of variance. The statistical significance of differences was determined using the Student's t-test or the Tukey–Kramer method. Differences were considered statistically significant at P < 0.05. Data are expressed as means ± SEM. We used our previously reported 3D culture system as the control culture (Figure 1A).22Katano T. Ootani A. Mizoshita T. Tanida S. Tsukamoto H. Ozeki K. Ebi M. Mori Y. Kataoka H. Kamiya T. Toda S. Joh T. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche.Biochem Biophys Res Commun. 2013; 432: 558-563Crossref PubMed Scopus (37) Google Scholar Cultured minced gastric tissue fragments from neonatal mice yielded expanding cystic structures termed gastrospheres (Figure 1B) that were surrounded by outer spindle cells (Figure 1, C and D). The outer lining of spindle cells in the matrix was positive for the myofibroblast marker α-SMA (Figure 1D). The previously established growth of gastric myofibroblasts was essential for the growth of gastrospheres. Gastric fragments without a sufficient number of outer myofibroblasts were not able to form gastrospheres. The placement of myofibroblasts in close proximity to the gastric epithelium in vitro mimicked the in vivo environment. α-SMA–positive spindle cells were present in the periglandular mesenchyme in vivo (Figure 1D). We established a mouse GMF cell line and used the cells at passages 4 to 10 in this study. Cultured spindle cells were positive for the myofibroblast marker α-SMA and vimentin (Figure 2, A–C), however, these cells were negative for cytokeratin, MUC5AC, HGM, MUC6, HIK1083, MUC2, CD10, chromogranin A, pepsinogen, and H+, K+-ATPase (data not shown). These results suggest that cultured cells had a similar expression pattern as gastric stromal myofibroblasts in vitro in the absence of epithelial cells. In our 3D culture system, cultured GMFs maintained similar immunohistochemical characteristics such as positive expression of α-SMA and vimentin (Figure 2, D–F). Next, to investigate the effect of GMFs on the growth of gastrospheres, we used the co-culture system. In the GMF co-culture system, growth of gastrospheres was more stable over the long term (Figure 3, A and B). On culture day 20, we measured the size and calculated the number of gastrospheres that had grown to >1 mm (Figure 3B). In the co-culture system and the control culture, the sizes of gastrospheres were 3.56 ± 0.41 mm and 2.13 ± 0.22 mm (P = 0.008), respectively, and the numbers were 13.67 ± 2.03 and 3.67 ± 0.67 (P = 0.009), respectively (Figure 3, C and D). Thus, GMFs significantly increased the growth and stability of gastrospheres. In both culture systems, the wall of the cultured gastrospheres consisted of a polarized epithelial monolayer with an apical, inner luminal surface, and a basal outer surface in close proximity to spindle-shaped cells and the collagen matrix. The cultured gastric epithelial cells showed a tall columnar shape, basally situated round nuclei, and clear cytoplasm that was filled with mucus (Figure 4A). The intracytoplasmic mucus stained strongly with periodic acid-Schiff (Figure 4B). In the control culture, HGM-positive epithelial cells and chromogranin A–positive cells were observed, indicating differentiation into gastric surface mucous cells and enteroendocrine cells, respectively. HIK1083-positive cells, indicating mucous neck cell differentiation, were scattered in the control culture on day 20. However, the epithelial cells were negative for the parietal cell marker H+/K+-ATPase and the chief cell marker pepsinogen in the control culture on day 20. In the GMF co-culture system, in addition to HGM-positive cells, HIK1083-positive cells were observed in the pit-like structures of gastrospheres, and less than 1% of the epithelial cells were positive for the enteroendocrine cell marker chromogranin A (Figure 4, A–C). Even in the co-culture system, H+/K+-ATPase– and pepsinogen-positive cells were not observed with immunohistochemistry. We next examined the effect of GMFs on the differentiation of the gastric epithelium as indicated by the expression of transcription factors. Muc5ac, Muc6, Pga5, PgC, Atp4a, and Chga expression was not significantly different between the co-culture system and control culture (P = 0.638, 0.521, 0.337, 0.315, 0.235, and 0.283, respectively) (Figure 4D). We confirmed Lgr5+ cells using RNA in situ hybridization. Lgr5+ brown dots appeared at the base of crypts in the small intestines and colons of adult mice, and at the bottom of the pyloric glands in neonatal mice (Figure 5, A–D). In the gastrospheres on culture day 20, Lgr5+ cells were detected in the epithelial layer (Figure 5E). On day 56, Lgr5+ cells were observed consistently in the epithelium of gastrospheres cultured in the GMF co-culture system (Figure 5E). In vitro and in vivo, Lgr5+ cells were observed only among epithelial cells, and not among mesenchymal myofibroblasts. We examined Lgr5+ epithelial cell populations in gastrospheres on day 20 in both culture systems. In the control culture system and the GMF co-culture, Lgr5+ epithelial cells were 7.15% ± 1.12% and 9.96% ± 3.61% (P = 0.499), respectively, of the cells present (Figure 5F). We next performed immunohistochemistry for Ki-67 to compare the proliferation ability of cultured epithelium in both culture systems (Figure 5G). The rate of Ki-67–positive cells in the GMF co-culture was higher than that in the control culture (11.838% ± 2.409% and 4.327% ± 1.619%, respectively; P = 0.032). The rate of Ki-67–positive cells was not significantly different between gastrospheres in the GMF co-culture system and gastric glands in vivo (11.838% ± 2.409% and 15.473% ± 0.314%, respectively; P = 0.173) (Figure 5G). These results suggest that GMFs allow maintenance of the cycle of gastric epithelial proliferation and differentiation, similar to that in vivo. To clarify the tissue-specific factors associated with the epithelial-mesenchymal interaction, we compared the expression levels of 46 genes between cultured GMFs and small intestinal myofibroblasts using real-time RT-PCR. Among various genes associated with the hedgehog signaling pathway, Notch, bone morphogenetic protein, fibroblast growth factor, Wnt, and homeobox genes, the mRNA levels of four genes were significantly different between GMFs and IMFs (Figure 6). The expression of Gas1 in GMFs was significantly higher than in IMFs (P = 0.044). The expression of Notch1 and Sox10 in IMFs was significantly higher than in GMFs (P = 0.014 and 0.049, respectively). Hoxc8 and Sox10 were identified as tissue-specific factors expressed exclusively in IMFs (P = 0.0003). Here, we report a method for long-term culture of primary mouse glandular stomach cells with mesenchymal myofibroblasts as the stem cell niche. By using our previous 3D culture method, this method resulted in increased gastric epithelial stability and induced multilineage differentiation. In the past, several researchers have reported methods for primary gastric culture.15Terano A. Ivey K.J. Stachura J. Sekhon S. Hosojima H. McKenzie Jr., W.N. Krause W.J. Wyche J.H. Cell culture of rat gastric fundic mucosa.Gastroenterology. 1982; 83: 1280-1291Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 16Sanders M.J. Amirian D.A. Ayalon A. Soll A.H. Regulation of pepsinogen release from canine chief cells in primary monolayer culture.Am J Physiol. 1983; 245: G641-G646PubMed Google Scholar, 18Chew C.S. Parietal cell culture: new models and directions.Annu Rev Physiol. 1994; 56: 445-461Crossref PubMed Scopus (44) Google Scholar, 19Ootani A. Toda S. Fuj
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