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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Departments of Clinical Laboratory Science (Y.H., T.Na., H.E., E.M., S.K.) and Gastroenterology and Hepatology (S.T., M.T., T.Ni., S.I., H.I., N.H.), Osaka University Graduate School of Medicine, Osaka, Japan; and Rinku General Medical Center, Izumisano Municipal Hospital, Osaka, Japan (S.K.)
Received for publication
January 23, 2008
Accepted
April 29, 2008.
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Abstract |
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) and clinical science (Baumgart and Sandborn, 2007), treatment of inflammatory bowel diseases (IBD) is far from satisfactory. Several studies suggest that immunological changes in lymphocytes are responsible for both the development and healing of IBD (Bouma and Strober, 2003). Myeloid cell function was also found to be important in a cohort of patients with Crohn's disease (Dieckgraefe and Korzenik, 2002). In contrast, emerging data suggest nonhematopoietic cells play a critical role in the development and healing of IBD (Olson et al., 2006). For example, the expression of Toll-like receptors in mucosal epithelia is altered in IBD (Cario and Podolsky, 2000). In addition, mucosal mesenchymal and endothelial cells play decisive roles through their interactions with immune cells (Fiocchi et al., 2006).
Other studies have shown that bone marrow-derived cells contribute to the healing of gastrointestinal wounds in humans (Okamoto et al., 2002; Matsumoto et al., 2005) and animals (Brittan et al., 2002, 2005; Komori et al., 2005a,b; Bamba et al., 2006; Hayashi et al., 2007; Khalil et al., 2007). Moreover, bone marrow-derived cells are recruited more efficiently to the injured gut than to a healthy gut, suggesting a rescue response for assisting with the recovery from damage (Komori et al., 2005a,b). Previous studies showed the transplantation of wild-type bone marrow in mutant mice lacking interleukin (IL)-10 apparently ameliorated an inflamed bowel (Bamba et al., 2006). Furthermore, the nonmyeloablative transplantation of immortalized cells in an experimental model of IBD promoted tissue regeneration (Khalil et al., 2007). However, it is still unclear what types of cells are able to exert these beneficial effects and thus which types would be most appropriate for IBD treatment.
In contrast to hematopoietic stem cells, bone marrow-derived mesenchymal stem cells (MSCs) are easily isolated by adherence to plastic dishes, and they are efficiently expandable in vitro (Peister et al., 2004). Due to their multipotency, MSCs are an attractive stem cell source in regenerative medicine. The aims of the present study were to examine the effects of topical implantation of bone marrow-derived MSCs on experimental colitis and to characterize phenotypes of the implanted MSCs. We used rats bearing colitis as an experimental model for investigating the beneficial effects of MSC implantation.
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Materials and Methods |
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Preparation of Bone Marrow-Derived Dish-Adherent Cells (Putative MSCs). Bone marrow cells were collected by flushing the bone shafts of the femurs and tibias of male rats with Medium 199 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal calf serum (JRH Biosciences, Lenexa, KS) and 1% antibiotics and antimycotics (Invitrogen) using a 21-gauge needle. After filtering the cells through a 50-µm nylon mesh and washing twice with medium, the bone marrow cells were cultured in 
-minimal essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 200 U/ml penicillin G sodium, and 200 U/ml streptomycin sulfate at a density of 1 x 108 cells per plastic dish (10 cm in diameter; Corning International, Tokyo, Japan). These cells made up the unselected bone marrow cells (BMCs). To isolate putative MSCs, after 24 h of culture, nonadherent cells were removed by washing the cells with phosphate-buffered saline (PBS), and adherent cells were maintained; the culture medium was replaced twice a week. The dish-adherent MSC population was expanded in 3 to 5 passages after the initial plating.
Characterization of the Putative MSCs. Expression of surface cell markers was examined by flow cytometry using FACScan (Immunocytometry Systems, San Jose, CA). Cells were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against CD31 (clone MCA1334FA; Serotec, Oxford, UK), rat CD34 (clone ICO-115; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), CD45 (clone OX-1; BD Biosciences, San Jose, CA), CD29 (clone HMβ1-1; BioLegend, San Diego, CA), CD90 (clone OX-7; BD Biosciences), -smooth muscle actin (-SMA: clone 1A4; Sigma-Aldrich, St. Louis, MO), and vimentin (clone V9; Santa Cruz Biotechnology, Inc.). Isotype-identical antibodies (clone 2E1; Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) served as controls.
The expression of IL-10 and growth factors typically found in MSCs was explored by reverse transcription-polymerase chain reaction (RT-PCR) using the GeneAmp PCR System 9600 (PerkinElmer Applied Biosystems, Roissy, France) and Ready-To-Go PCR Beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK). To monitor cDNA synthesis efficiency, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The PCR primers used for detecting rat vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1, IL-10, and GAPDH cDNA are shown in Table 1. PCR products were electrophoresed in 2.0% agarose gels containing ethidium bromide in 1x Tris acetate-EDTA buffer.
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To investigate whether MSCs produced VEGF, we measured VEGF levels in the MSC-conditioned medium (1 x 106 cells in 10-cm dish cultured for 48 h). VEGF and TGF-β1 were measured using an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's protocol (VEGF immunoassay and TGF-β1 immunoassay; R&D Systems, Minneapolis, MN).
Multipotent MSCs are able to differentiate into osteocytes, adipocytes (Prockop, 1997; Pittenger et al., 1999), and other types of cells. The differentiation potential of bone marrow-derived dish-adherent cells (passage 5) into osteocytes or adipocytes was assessed using differentiation-induction media (KE-200 and KE-300; DS Pharma Biomedical Co., Ltd., Osaka, Japan) according to the manufacturer's protocols. The medium was changed three times per week for 21 days. For assessing osteogenesis, the cells were fixed with 10% formalin for 20 min at room temperature and stained with Alizarin Red, pH 4.1 (Sigma-Aldrich). For assessing adipogenesis, the cells were fixed with 10% formalin for 20 min at room temperature and stained with 0.5% oil red O (Sigma-Aldrich) in methanol (Sigma-Aldrich) for 20 min at room temperature.
Induction of TNBS Colitis. Colitis was produced in 7-week-old Sprague-Dawley rats using the method of Uchida and Mogami (2005), with minor modifications. In brief, the rats were laparotomized under sevoflurane anesthesia (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan). The proximal colon was clamped with ringed forceps that had a 10-mm inner diameter. An ethanol solution (35%; 0.2 ml) containing 0.15 M TNBS (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was injected into the proximal colon lumen via a 29-gauge needle. The clamp was maintained for 2 min to allow colon injury to develop (Fig. 1).
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), to enable tracing the cells in vivo. The PKH67-labeling procedure for adult somatic stem cells has been described previously in detail (Askenasy and Farkas, 2002; Grøgaard et al., 2007). In brief, 1 x 107 cells were incubated in 1 ml of diluent with PKH67 for 5 min as suggested by the manufacturer. The cells were collected by centrifugation (400g for 10 min), resuspended, and washed twice with PBS. The recovery rate of this procedure was 80 to 90% viable cells. Immediately after the TNBS-induced colon injury, the rats were randomized, and they received injections with 1 x 107 putative MSCs in 100 µl of PBS per animal or with 100 µl of PBS alone. Submucosal injections were given into the colonic wall at three points surrounding the damaged area (Fig. 1). In addition, another group of rats was randomized into two groups, and they received injections with 1 x 107 unselected BMCs in PBS (100 µl) or with 100 µl of PBS alone. The abdomen was closed and the animal was allowed to recover from the procedure with free access to tap water and standard pellet chow.
Assessment of TNBS-Induced Colonic Injury. The systemic influence of colitis was first evaluated by weighing each animal daily. On day 6, the rats were anesthetized and sacrificed for macroscopic and microscopic analyses of the TNBS-induced colonic injury. The chest and abdominal wall were opened with a midline incision, and the inferior vena cava was cut for exsanguination. The organs were perfused via the heart with PBS and 4% paraformaldehyde (Sigma-Aldrich) to flush out blood cells. The colon was opened, and the size of the injury was measured. The injured colon section was excised, prefixed in 4% paraformaldehyde, and placed on ice for 3 h. It was then dehydrated through graded sucrose washes for 24 h, embedded in Tissue-Tek OCT compound (Sakura Finechemical Co., Tokyo, Japan), and frozen in liquid nitrogen. Semithin sections were prepared and stained with hematoxylin and eosin for histopathological assessment.
For immunohistochemical analysis, cryostat sections (5 µm in thickness) were incubated at 37°C for 1 h in 3% normal goat serum to prevent putative nonspecific binding to mouse immunoglobulins. Then, the sections were incubated at 4°C for 24 h with primary antibodies against vimentin [clone V9; YLEM, Avezzano (AQ), Italy], -SMA (clone 1A4; Dako UK Ltd., Ely, Cambridgeshire, UK), desmin (clone RD-301; Santa Cruz Biotechnology, Inc.), von Willebrand factor (A0082; Dako UK Ltd.), Ki-67 (YLEM), VEGF (clone A-20; Santa Cruz Biotechnology, Inc.), and TGF-β1 (clone TB21; Oxford Biotechnology, Co., Ltd., Oxford, UK). After three washes for 5 min each in PBS, the sections were incubated with the appropriate secondary antibodies labeled with rhodamine red. To visualize cell nuclei, the sections were stained with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA), and then they were observed under a fluorescence microscope (Nikon Eclipse TE2000-U; Nikon, Tokyo, Japan) or a confocal laser-scanning microscope with appropriate filters (LSM510; Carl Zeiss, Oberkochen, Germany). The colon wall was examined except for the granulation tissue, which was too fragile for processing. All images were captured by a digital imaging system.
Statistical Analysis. Flow cytometry, RT-PCR, and differentiation assays were performed at least in triplicate, and typical results were presented. Parametric data are expressed as mean ± S.D., and they were analyzed by a two-tailed Student's t test or analysis of variance followed by multiple comparisons of the means. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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MSCs secrete a variety of growth factors and cytokines (Liu et al., 2006). The RT-PCR study demonstrated that the cells at passage 5 expressed VEGF and TGF-β1 mRNAs (Fig. 2). However, the cells did not express IL-10 mRNA (Fig. 3). After 48 h in culture, MSCs secreted more VEGF and TGF-β1 than unselected BMCs. The phenotypes of the bone marrow-derived dish-adherent MSCs are summarized in Table 2.
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Effects of MSC Implantation on Development and Healing of TNBS-Induced Colitis. As a preliminary experiment, we examined the effects of i.v. administration of PKH67-labeled MSCs (1 x 107 cells) on TNBS-induced colitis in rats. However, i.v. administration of MSCs did not mitigate the colonic damage and body weight loss induced by TNBS. Furthermore, PKH67-labeled MSCs accumulated in lung rather than in colonic tissues, including those surrounding the TNBS injury (data not shown).
As expected, body weight increased gradually in untreated rats. In contrast, rats treated with luminal TNBS and submucosal PBS injections (TNBS + PBS) were exhausted, and they exhibited a significant loss of body weight. Significant and continuous weight loss was also observed in rats that received luminal TNBS and implantation of unselected BMCs (TNBS + BMCs). However, the rats that received luminal TNBS and MSCs implantation (TNBS + MSCs) exhibited a transient loss of body weight followed by a recovery. The body weight was significantly higher in the TNBS + MSCs group than in the TNBS + PBS group on days 3 to 6 (Fig. 4A). It is interesting to note that transplantation of freshly isolated, unselected BMCs (1 x 107 cells) did not mitigate the body weight loss induced by TNBS injections (Fig. 4B).
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Histological analysis showed that, as reported originally (Uchida and Mogami, 2005), TNBS caused ulcerative colon injury penetrating toward the proper muscle layer. On day 3, the lesions were characterized by edema, epithelial exfoliation, and infiltration of leukocytes. The ulcer margins were clear, and they did not yet develop immature epithelium in any of the groups. On day 6, the ulcer margins had an extension of regenerated epithelia in all the groups. The proper muscle layer was disrupted at the site of ulceration throughout the study period. However, on day 6, the depth of the tissue defect was shallower in rats with MSC implantation than in controls (Fig. 6, a and b).
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-SMA and the smooth muscle marker desmin (Fig. 7A). Furthermore, PKH67-positive, -SMA-positive MSCs were spindle-shaped, indicating that a minor population of MSCs had differentiated into myofibroblasts. The percentages of transplanted MSCs that expressed vimentin, -SMA, and desmin were 47.2 ± 14.6, 6.1 ± 3.2, and 7.3 ± 4.9, respectively (Fig. 7B). These results suggest that MSCs were able to differentiate into interstitial lineage cells, fibroblasts, and myofibroblasts. It should be noted that the colon also contained PKH67-negative host cells expressing vimentin, -SMA, or desmin. Moreover, there was neither endothelial cell positive for PKH67 and von Willebrand factor nor proliferating cell positive for PKH67 and Ki-67 in the colon (data not shown). Furthermore, when they were implanted into the normal colon (non-TNBS), MSCs partly differentiated into colonic interstitial lineage cells 6 days after the implantation (Fig. 8). These results suggested that the differentiation of engrafted MSCs in the colon did not depend on the colonic inflammation.
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We also found expression of VEGF and TGF-β1 in colon mucosa after MSC implantation. The RT-PCR study showed that the expression of VEGF and TGF-β1 mRNAs in the colonic tissues was not apparently different between the TNBS + PBS group and TNBS + MSCs groups 6 days after the implantation (data not shown). However, VEGF and TGF-β1 immunostaining was observed in PKH67-positive MSCs surrounding the lesion area on day 3 (Fig. 9) and day 6 (Fig. 10A), suggesting the implanted MSCs secreted VEGF and TGF-β1 locally. The percentages of MSCs that expressed VEGF and TGF-β1 were 27.6 ± 16.6 and 26.5 ± 15.5, respectively, 6 days after the implantation (Fig. 10B). It is noteworthy that some MSCs expressing VEGF or TGF-β1 were not necessarily thin-spindle-shaped. Rather, the majority of MSCs expressing VEGF or TGF-β1 were oval- or thick-spindle-shaped. VEGF and TGF-β1 were also expressed in PKH67-negative, spindle-shaped cells; these results indicated that host-derived interstitial lineage cells might also be sources of TGF-β1 and VEGF in the colon.
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Discussion |
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-SMA (a marker for myofibroblast/smooth muscle) or desmin (type III intermediate filaments: a marker for myocytes, smooth muscles, and other muscle cells). These results suggest that MSCs had a tendency to differentiate into interstitial or stromal lineage cells. The labeled cells were well distributed within the site of injection and did not form tumors or capsules. Therefore, the implantation of MSCs seemed to be safe; however, the long-term fate of the engrafted MSCs remains to be investigated.
The size of the TNBS-induced colon injury by day 3 was comparable between the rats that received MSC implantation and those given PBS. However, by day 6, the injury was significantly smaller in the TNBS + MSCs group than the TNBS + PBS group. Histological analyses also showed that by day 6, the depth of the injury was significantly shallower in the TNBS + MSCs group than in the TNBS + PBS group. These results show that topical implantation of MSCs accelerated the healing of a colon injury. In an earlier period, (i.e., on day 3 after the colitis induction) engrafted MSCs also differentiated into colonic interstitial lineage cells and produced VEGF and TGF-β1. These results suggest that the implantation at least partially ameliorated TNBS-induced colonic inflammation.
A previous study reported that transplantation of wild-type bone marrow cells ameliorated spontaneous colitis in IL-10 null mice (Bamba et al., 2006), suggesting an important role of this cytokine in the development and healing of colon inflammation. However, in the present study, MSCs cultured in vitro did not express IL-10 mRNA. Therefore, it is unlikely that the implantation accelerated healing of the TNBS-induced colon injury by providing IL-10. In contrast, our RT-PCR and ELISA results clearly demonstrated that MSCs cultured in vitro expressed and secreted both VEGF and TGF-β1. Furthermore, fluorescence immunohistochemistry showed that the engrafted MSCs also expressed these growth factors in the injured colon in vivo. TGF-β1 and VEGF are known to be produced in various types of cells, and to play important roles in gastrointestinal wound healing. TGF-β1 has potent effects on gastrointestinal mucosal integrity, wound repair (Beck et al., 2003), angiogenesis (Daniel and Abrahamson, 2000), and the regulation of acquired (Letterio and Roberts, 1998) and innate immunity (Meneghin and Hogaboam, 2007). In human IBD, TGF-β1 expression was reported to be enhanced in gut mucosa (Babyatsky et al., 1996). In a mouse model of IBD, the loss of TGF-β signaling contributed to intestinal injury (Hahm et al., 2001). It is interesting to note that inflammatory cell infiltration was smaller in the TNBS + MSCs group than in the TNBS + PBS group, suggesting anti-inflammatory influences of the MSC engrafts. Ryan et al. (2005, 2007) reported that MSCs interfered with dendritic cells and T-cell function and generated a local immunosuppressive microenvironment by modulating interferon-
, proinflammatory cytokine.
TGF-β1 is also reported to activate fibroblasts and other cells to promote wound contraction (Wynn, 2007). In that study, MSCs showed a tendency to differentiate into fibroblasts, myofibroblasts, and other interstitial lineage cells, when they were incubated in vitro. However, in the present study, a majority the engrafted MSCs and their descendants were positive in vivo for only vimentin, and not for -SMA or desmin. Therefore, our results suggest that most MSCs may not differentiate to myofibroblasts or to smooth muscle cells within 6 days after implantation. Thus, the contribution of the implanted MSCs in wound contraction seemed to be minimal. However, MSC-derived TGF-β1 may play a role in wound contraction via its effects on host myofibroblasts and other cells; this remains to be investigated in a long-term study.
VEGF is a growth factor that stimulates recruitment of both leukocytes and vascular endothelial cells. The importance of VEGF in wound healing in the gut has been described in detail (Tarnawski, 2005). Therefore, our results suggest that MSCs may contribute, at least in a part, to the healing of injured colonic mucosa by secreting TGF-β1 and VEGF. Consistent with this notion, a potent therapeutic effect of MSCs, probably dependent on TGF-β1 and VEGF, was reported recently in a study focused on chronic inflammation of the kidneys (Kunter et al., 2006).
A growing body of data suggests that bone marrow contains adult somatic stem cells that are involved in the healing process of gut injury. Bone marrow-derived cells are temporally and efficiently recruited into gastrointestinal tissue during the healing process of chemical injury (Komori et al., 2005a,b). The involvement of the bone marrow-derived cells into gastrointestinal tissue reduces after the damage was completely healed, suggesting that the bone marrow-derived cells participate positively in wound healing. Brittan et al. (2005) reported that bone marrow cells were involved in neovasculogenesis in the colon after TNBS-induced colitis in mice. Furthermore, Khalil et al. (2007) reported that i.v. infusion of CD34-negative cells immortalized with simian virus 40 large-T antigen promoted tissue regeneration and involved in neovasculogenesis in murine dextran sulfate sodium-induced colitis (Khalil et al., 2007). However, in the present study using nonimmortalized MSCs, we did not find any involvement of MSCs in neovasculogenesis 6 days after the induction of TNBS-induced colitis. These results in rats are consistent to the findings of Sato et al. (2005) who reported that there was no evidence of differentiation of human MSCs into CD31-positive endothelial cells in the rat liver. Although the reasons for these discrepancies remain unclear, the discrepancy between these studies might be due to differences in the methods used to transplant stem cells, differences in the experimental animals used, differences in the phenotype of stem cells used and different methods used for tissue analyses.
Finally, the present data demonstrate that MSCs can be engrafted safely and successfully by topical injection. The preliminary study showed that nonimmortalized CD29-positve, CD90-positve, CD31-negative, CD34-negative, and CD45-negative MSCs were trapped mainly in lung, and they were hardly found in the injured colon. Furthermore, the i.v. administration of 1 x 107 MSCs was insufficient to accelerate healing of TNBS-induced injury, whereas the topical implantation of the same number of MSCs was sufficient to obtain the beneficial effects. The topical injection enables to deliver larger amount of nonimmortalized MSCs than i.v. administration proposed by Khalil et al. (2007). These findings are clinically relevant, because the gastrointestinal tract is easily accessible with endoscope technology. Active IBD, i.e., Crohn's disease and ulcerative colitis, often displays deep lesions that can be identified under colonoscopy or enteroscopy. Endoscopic injection has been used in hemostatic treatments against acute gastrointestinal bleeding and in the nonsurgical removal of colon adenomas and early stage carcinomas (Sivak, 2000). We have shown that topical injection can be applied to implantation of MSCs into a target area.
In conclusion, we have isolated and expanded MSCs in vitro and successfully implanted them into inflamed gut tissue in rats in vivo. The engrafted MSCs survived and accelerated healing of TNBS-induced colitis. After the implantation, the MSCs became potential sources of VEGF and TGF-β1, angiogenic and immunomodulating factors, in colon tissues. Minor populations of the MSCs differentiated into myofibroblasts, fibroblasts, and other interstitial lineage cells; these may also have participated in healing TNBS-induced colitis. Thus, the topical implantation of MSCs is a potentially safe and useful strategy for the treatment of IBD.
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Footnotes |
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ABBREVIATIONS: IBD, inflammatory bowel disease; IL, interleukin; MSC, mesenchymal stem cell; BMC, bone marrow-derived cell; PBS, phosphate-buffered saline; -SMA, -smooth muscle actin; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VEGF, vascular endothelial growth factor; TGF, transforming growth factor; ELISA, enzyme-linked immunosorbent assay; TNBS, 2,4,6-trinitrobenzene sulfonic acid; DAPI, 4',6-diamidino-2-phenylindole.
Address correspondence to: Dr. Shingo Tsuji, Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: stsuji{at}gh.med.osaka-u.ac.jp
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