AJP - GI Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 294: G778-G786, 2008. First published January 17, 2008; doi:10.1152/ajpgi.00468.2007
0193-1857/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/G778    most recent
00468.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, Y.
Right arrow Articles by Kawano, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, Y.
Right arrow Articles by Kawano, S.

MUCOSAL BIOLOGY

Topical transplantation of mesenchymal stem cells accelerates gastric ulcer healing in rats

Yujiro Hayashi,1 Shingo Tsuji,2 Masahiko Tsujii,2 Tsutomu Nishida,2 Shuji Ishii,2 Hideki Iijima,2 Toru Nakamura,1 Hiroshi Eguchi,1 Eiji Miyoshi,1 Norio Hayashi,2 and Sunao Kawano1,3

1Department of Clinical Laboratory Science and 2Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita; and 3Izumisano Municipal Hospital, Izumisano, Osaka, Japan

Submitted 10 October 2007 ; accepted in final form 15 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mesenchymal stem cells (MSCs), a subpopulation of adult somatic stem cells, are an attractive stem cell source in regenerative medicine because of their multipotentiality. We examined the effects of MSC transplantation on gastric ulcer healing. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in culture, were characterized by flow cytometry and reverse transcription-polymerase chain reaction. Gastric ulcers were induced by serosal application of acetic acid on the anterior wall of the stomach in female rats. Either MSCs (labeled with PKH67; 1x107 cells) or vehicle was injected into the gastric wall surrounding the ulcer. The healing process of the ulcer and the influence of anti-vascular endothelial growth factor (VEGF) antibody were examined. CD29-positive, CD90-positive, CD34-negative, and CD45-negative MSCs expressed mRNAs for VEGF and hepatocyte growth factor (HGF). The MSCs were transplantable to the gastric tissue surrounding the ulcer, where a majority of the engrafted cells were positive for vimentin. The transplantation significantly accelerated gastric ulcer healing compared with controls. The engrafted MSCs also expressed VEGF and HGF. Administration of anti-VEGF neutralizing antibody dose dependently reduced the MSC-induced promotion of ulcer healing. In conclusion, MSC transplantation accelerated gastric ulcer healing, possibly through the induction of angiogenesis in the gastric mucosa via the secretion of VEGF. The beneficial effects of MSCs might be mediated not only by their differentiation into gastric interstitial cells, but also by their ability to supply angiogenic factors.

bone marrow; mesenchymal stromal cells

GASTRIC ULCER HEALING IS A complex process that involves inflammation, reepithelialization, granulation tissue formation, angiogenesis, interactions between various cells and the matrix, and scar formation (18, 19, 26, 29). Several groups, including our own, have reported that bone marrow-derived adult somatic stem cells contribute to the formation of gastrointestinal tissues, including vascular endothelial cells, epithelium, and interstitial cells, and have an important role in healing gut injuries (1, 3, 4, 8, 10, 11, 14, 20).

Mesenchymal stem cells (MSCs) are a subpopulation of multipotent adult somatic stem cells that reside within the bone marrow and a wide variety of organs. In contrast to hematopoietic stem cells, bone marrow-derived MSCs are adherent to plastic dishes and can be expanded in vitro (21). MSCs differentiate into osteocytes, chondrocytes, adipocytes (22, 23), vascular endothelial cells (24), and other types of cells. Therefore, MSCs are an attractive stem cell source in regenerative medicine because of their multipotentiality. MSCs directly injected into an injured heart or liver induce tissue regeneration and improve organ function (17, 25). There is little information, however, about the therapeutic effects of MSCs in gastrointestinal disorders.

In the present study, we examined whether topical transplantation of MSCs accelerated gastric ulcer healing. We also explored the mechanisms underlying the effects of the transplanted MSCs on gastric ulcer healing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. Male and female Sprague-Dawley rats were purchased from Japan SLC (Shizuoka, Japan). All animal studies were reviewed and approved by the institutional committee on the use and care of animals at Osaka University Graduate School of Medicine.

Expansion of bone marrow-derived dish-adherent cells. Bone marrow cells were collected by flushing the bone shafts of the femurs and tibias of male rats with a 21-gauge needle containing medium 199 (Invitrogen, San Diego, CA) supplemented with 5% fetal calf serum (JRH Bioscience, Lenexa, KS) and 1% antibiotics and antimycotics (Invitrogen). After the cells were sieved through 50-µm nylon mesh and washed twice with the medium, the bone marrow cells were cultured in {alpha}-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 1x108 cells per plastic dish (10 cm in diameter; no. 430167; Corning International, Tokyo, Japan). Nonadherent hematopoietic cells were removed, and the medium was replaced. The dish-adherent MSC population was expanded within three to five passages after the cells were first plated.

Flow cytometry. Flow-cytometric analysis was performed by using a FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The cells were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against rat CD34 (clone ICO-115, Santa Cruz Biotechnology, Santa Cruz, CA), CD45 (clone OX-1, BD Biosciences, San Jose, CA), CD29 (clone HMβ1-1, BioLegend, San Diego, CA), and CD90 (clone OX-7, BD Biosciences). The cells were fixed in 1% paraformaldehyde and incubated with an fluorescein isothiocyanate-conjugated monoclonal antibody against {alpha}-smooth muscle actin ({alpha}-SMA: clone 1A4, Sigma) and vimentin (Clone V9, Santa Cruz). Isotype-identical antibody (clone 2E12, Medical & Biological Laboratories, Nagoya, Japan) served as negative controls.

RT-PCR. To determine whether the cells expressed growth factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in culture, we performed reverse transcription-polymerase chain reaction (RT-PCR) assay using GeneAmp PCR System 9600 (Perkin-Elmer Applied Biosystems, Roissy, France) and Ready-To-Go PCR Beads (Amersham Pharmacia Biotech, Piscataway, NJ). To monitor cDNA synthesis efficiency, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR primers for rat VEGF (515 bp) and HGF (580 bp) cDNA were as follows: VEGF (rat) forward 5'-AGT CTT GCC AAT GTG GAC TC-3'; VEGF (rat) reverse 5'-GGC AGT GGA TTC TCA TCT TG-3'; HGF (rat) forward 5'-TCC ATT CAC TTG CAA GG-3'; HGF (rat) reverse 5'-TAG CAC CAT GGC CTC GG-3'; GAPDH (rat) forward 5'-TCC CTC AAG ATT GTC AGC AA-3'; GAPDH (rat) reverse 5'-AGA TCC ACA ACG GAT ACA TT-3'. The amplification conditions were 34 cycles for VEGF, 32 cycles for HGF, and 28 cycles for GAPDH. PCR products were electrophoresed in 2.0% agarose gels containing ethidium bromide in 1 x Tris-acetate-EDTA (TAE) buffer and photographs were taken.

ELISA. To investigate whether MSCs produce VEGF, we measured VEGF levels in the MSC culture supernatant (1x106 cells in 10-cm dish cultured for 48 h). VEGF was measured with an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's protocol (VEGF Immunoassay, R&D Systems, Minneapolis, MN).

Evaluation of differentiation of bone marrow-derived dish-adherent cells. The differentiation potential of bone marrow-derived dish-adherent cells (passage 5) into osteocytes or adipocytes was evaluated by use of differentiation-induction media purchased from Dainippon-Sumitomo Pharma (Osaka, Japan) according to the manufacturer's directions. Media inducing differentiation toward the other lineages were unavailable and were not applied. The medium was changed three times per week for 21 days. To evaluate osteogenesis, the cells were fixed with 10% formalin for 20 min at room temperature and stained with alizarin red, pH 4.1 (Sigma-Aldrich Chemical, St. Louis, MO). To evaluate adipogenesis, the cells were fixed with 10% formalin for 20 min at room temperature and stained with 0.5% Oil Red O (Sigma) in methanol (Sigma) for 20 min at room temperature. As the control, the bone marrow-derived dish-adherent cells were incubated without any differentiation-induction stimuli and stained with alizarin red or Oil Red O.

Embryonic stem cells have a potential to develop teratomas and even teratocarcinomas when they are transplanted in an undifferentiated state into another individual. (16) To explore their normality, we examined whether MSCs have tumorigenic potential. Four female Sprague-Dawley rats were inoculated subcutaneously with PKH67-labeled MSCs (1x107) per animal. One month after the inoculation, the rats were killed and the inoculated tissues were subjected to macroscopic and histological examination.

Induction of gastric ulcers. Acetic acid ulcers were produced in female rats aged 7 wk according to the method of Okabe and Amagase (19). In brief, the rats were laparotomized under sevoflurane anesthesia (Maruishi Pharmaceutical, Osaka, Japan). The anterior wall of the gastric angle was touched with 100% acetic acid in a cotton-plugged plastic tube, 6 mm in diameter, for 60 s.

Cell transplantation. As a preliminary experiment, we examined the influence of the number of cells transplanted on ulcer healing. Because the effect of 1 x 106 cells was modest, we used 1 x 107 of the bone marrow-derived dish-adherent cells for transplantation. Before the cell transplantation, the bone marrow-derived dish-adherent cells were labeled by the fluorescent cell linker PKH67 (Sigma). PKH dyes were initially developed by Horan and colleagues (15) to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. This dye has a long aliphatic carbon tail and a strong and stable fluorescence with excitation wavelength of 490 nm and emission of 504 nm. PKH67 is less immunogenic and less toxic than genetically introduced markers such as β-galactosidase. The dye does not cause extracellular leakage and does not influence cell proliferation. Stability of PKH67 has been established by studies tracking a wide range of cells including proliferative lymphocytes (30), cancer cells (5), and adult somatic stem cells in vivo (2, 7, 25a, 32). Consequently, it is reasonable to use the membrane dye for the analyses of MSCs in gastrointestinal tract in rats as well as in vivo tracking studies after autologous transplantation of moderate length.

Immediately after inducing the gastric ulcer, we injected a total of 1 x 107 cells/200 µl in phosphate-buffered saline (PBS), or PBS alone, subserosally into the gastric wall at five different points surrounding the ulcer. The animals were allowed to recover from the procedure with free access to tap water and standard pellet chow.

The effects of bone marrow cells (BMCs), freshly prepared from bone marrow but not selected by dish adherence, on ulcer healing were also compared with PBS injection. A total of 1 x 107 cells of BMCs in 200 µl PBS, or PBS alone, was subserosally injected at five different points surrounding the ulcer.

Tissue preparation and immunohistochemistry. At 3, 6, and 9 days after the ulcer induction, the rats were anesthetized and killed for macroscopic and microscopic analyses of ulcer healing. The chest and abdominal wall were opened with a midline incision and the inferior vena cava was cut. The organs were perfused via the heart with PBS and 4% paraformaldehyde to flush out blood cells. The stomach was opened along the greater curvature and the size of the ulcer was measured. The ulcer index was calculated as the product of the longest and shortest diameters of each lesion. The gastric tissue was fixed in 4% paraformaldehyde on ice for 3 h, dehydrated through graded sucrose washes for 24 h, embedded in Tissue-Tek OCT compound (Sakura Finechemical, Tokyo, Japan), and frozen in liquid nitrogen. For immunohistochemical analysis, cryostat sections (5-µm thick) were incubated at 37°C for 1 h in 3% normal goat serum to prevent putative nonspecific binding to mouse immunoglobulins. The sections were then incubated in primary antibodies against vimentin (clone V9, YLEM), {alpha}-SMA (clone 1A4, Dako), desmin (clone RD-301, Santa Cruz), von Willebrand factor (A0082, Dako), Ki-67 (YLEM), VEGF (clone A-20, Santa Cruz), and the {alpha}-subunit of HGF (HGF-{alpha}: IBL, Takasaki, Gumma, Japan) at 4°C for 24 h. After three washes for 5 min each in PBS, the sections were incubated in the appropriate secondary antibodies and labeled with rhodamine red. To examine specificity of immunostaining, other sections were incubated in rhodamine red-labeled nonimmunized IgG. To visualize nuclei under a fluorescence microscope, the sections were stained with VECTASHIELD with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The sections were observed under a fluorescence microscope (Nikon Eclipse TE2000-U, Tokyo, Japan) or a confocal laser-scanning microscope with the appropriate filters (Carl Zeiss LSM510, Oberkochen, Germany). The gastric wall was examined except for the granulation tissue, which was too fragile for this process. All images were captured with a digital imaging system.

Immunoneutralization of VEGF. Acetic acid-induced ulcers were induced at the anterior wall of the gastric angle, and the bone marrow-derived dish-adherent cells were transplanted in rats as described above. On days 3, 4, and 5 following the ulcer induction, the rats were injected intraperitoneally with 1, 3, or 10 µg of goat polyclonal VEGF neutralizing antibody (R&D Systems) or with normal goat immunoglobulin of the same isotype (R&D Systems). Rats were killed on day 6 and the stomachs were excised and used to assess the involvement of VEGF in gastric ulcer healing accelerated by cell transplantation.

Statistical analysis. Flow cytometry, RT-PCR, and differentiation assays were performed in triplicate at least, and typical results were demonstrated. Parametric data are shown as means ± SD. Statistical significance was determined by a two-tailed Student's t-test. A P value less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the bone marrow-derived dish-adherent cells. The bone marrow-derived cells isolated by dish adherence and expanded as described above were characterized by flow cytometry, differentiation assay, RT-PCR, and ELISA. More than 94% of the cells expressed CD29 and CD90 (Fig. 1). In contrast, there were virtually no CD34-positive immature hematopoietic cells and CD45-positive leukocyte common antigen in the population.


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 1. Flow-cytometric analysis of the bone marrow-derived dish-adherent cells at passage 5. In general, more than 94% of the cells expressed CD29 and CD90, whereas they were negative for CD34 and CD45. Broken lines are the results of staining with isotype-identical antibody as a negative control.

 
We examined the multipotentiality of the cells. The cells at passage 5 readily differentiated into Oil Red O-positive adipocytes or alizarin red S-positive mineralizing cells when incubated in the respective differentiation media for 21 days (Fig. 2). The cells incubated without any differentiation stimuli were negative for either Oil Red O or alizarin red S, suggesting that they were not contaminated with either adipocytes or osteocytes. These results indicated that the cells obtained from bone marrow, isolated by dish adherence, and expanded have the phenotype of immature nonhematopoietic stromal cells. Although a minor population expressed {alpha}-SMA, the cells maintained the ability to differentiate toward adipocytes or mineralizing cells. In addition, the morphology of MSCs at passage 5 was quite similar to that of MSCs at passage 3. The cells maintained spindle-shaped appearance, which was a typical phenotype of MSCs (21). FACS analysis showed that only minor populations of the cells expressed {alpha}-SMA (<20%) and vimentin (<10%) after five passages. Therefore, the cells isolated and expanded appeared to be multipotent MSCs derived from bone marrow.


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 2. Differentiation of the dish-adherent cells into adipocytes and osteoblasts/osteocytes. Under adipogenic conditions, the cells differentiated into adipocytes, which were positively stained by Oil Red O staining (bar = 50 µm) (A). The cells prior to adipogenic conditions that stained for Oil Red O were negligible (B). Under osteoblastic conditions, mesenchymal stem cells (MSCs) at passage 5 were induced to differentiate into osteoblasts or osteocytes, which were positively stained with alizarin red S (C). The cells prior to osteoblastic conditions that stained for alizarin red S were negligible (D) (bar = 50 µm). The images are representative of 3 independent experiments with similar results.

 
MSCs appeared to be normal and they did not form any tumors or teratomas 1 mo after the subcutaneous inoculation.

MSCs secrete a variety of growth factors (17). In the present study, the RT-PCR results demonstrated that the cells at passage 5 expressed mRNAs for VEGF, a potent angiogenic factor, and for HGF, a potent epithelial mitogen (Fig. 3A). After 48 h of culture, MSCs secreted more VEGF than BMCs (Fig. 3B).


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 3. Expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in the dish-adherent putative MSCs in vitro. A: expression of VEGF and HGF mRNAs in the bone marrow-derived dish-adherent cells after 5 passages in vitro. The RT-PCR results indicated that the cells expressed mRNAs for VEGF and HGF. M, molecular markers; lane 1, H2O; lane 2, VEGF; lane 3, HGF; lane 4, GAPDH. B: ELISA assay for VEGF secreted from cultured MSCs (1x106 cells) compared with total bone marrow cells (BMCs; 1x106 cells). BMCs were freshly isolated unselected cells. Data from 6–8 dishes are expressed as means ± SD. *P < 0.05 vs. BMCs.

 
Transplantation of MSCs accelerated gastric ulcer healing. Transplantation of MSCs significantly accelerated the healing of acetic acid-induced gastric ulcers compared with nontransplanted controls (vehicle injection) on day 6 and on day 9, but not on day 3 (Fig. 4A). On day 6, the ulcer index was 38.2 ± 18.4 mm2 in the vehicle-treated controls and 12.3 ± 10.7 mm2 (P < 0.001) in the MSC-transplanted group. On day 9, the ulcer was mostly closed in rats that received MSC transplantation (ulcer index: 1.5 ± 2.07 mm2), whereas the ulcer remained open (ulcer index: 6.73 ± 5.37 mm2; P < 0.05) in the vehicle-treated controls (Fig. 4). Microscopic analysis indicated that penetrating ulcers had successfully developed after serosal application of acetic acid in both groups by day 3. On day 3, the ulcer margin had not yet developed immature epithelium in either group. On days 6 and 9, however, the ulcer margin contained regenerated epithelia in both groups. The muscularis propria was disrupted at the site of ulceration throughout the study period. On days 6 and 9, however, the size of the tissue defect was significantly smaller in the MSC-transplanted rats compared with the controls. On the other hand, transplantation of freshly isolated, unselected BMCs (1x107 cells) did not accelerate the healing of acetic acid-induced gastric ulcers compared with vehicle injection on day 3, on day 6 and on day 9 (Fig. 4B).


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. Effects of transplantation of the putative MSCs (A) and freshly isolated, unselected BMCs (B) in the healing process of the acetic acid-induced ulcers in rats. Rats with gastric ulcers were killed 3, 6, and 9 days after the ulcer induction, and the longest and shortest diameters of the ulcer were measured under macroscopy. The ulcer index was calculated as the product of the longest and shortest diameters. Healing of acetic acid-induced ulcers was significantly accelerated by transplantation of MSCs compared with the control group (PBS injection) on day 6 and day 9, but on not day 3. Transplantation of BMCs did not accelerated significantly the healing of gastric ulcers compared with PBS injection on day 3, on day 6, and on day 9. Data from 4–12 rats are expressed as means ± SD. *P < 0.05 vs. control group treated with the vehicle (PBS) only; N.S, not significant.

 
Fluorescence microscopy revealed a heterogeneous distribution of the green fluorescent (PKH67)-labeled MSCs within the gastric tissue surrounding the ulcer. For this reason, it was impractical to count the number of PKH67-positive MSCs retained within the ulcer margin, although the percentage of subpopulations to total MSCs could be analyzed by immunostaining. On day 6 after the transplantation, MSCs were engrafted into the gastric submucosal layers where the cells were supposed to be injected (Fig. 5). Immunofluorescence microscopy indicated that the transplanted MSCs were partially positive for an interstitial marker vimentin, and a small portion of transplanted MSCs were positive for a smooth muscle cell/myofibroblast marker, {alpha}-SMA, and a smooth muscle marker, desmin (Fig. 6A–I). Furthermore, PKH67-positive, {alpha}-SMA-positive MSCs were spindle shaped, indicating that these cells differentiated into gastric myofibroblasts. The percentages of transplanted MSCs that expressed vimentin, {alpha}-SMA, and desmin were 56.5 ± 10.3, 5.1 ± 1, and 7.9 ± 6.2%, respectively (Fig. 6J). These results suggest that MSCs are able to differentiate into interstitial lineage cells, such as myofibroblasts and smooth muscle cells. Nevertheless, most of the transplanted MSCs are not yet differentiated terminally into myofibroblasts or smooth muscle cells. There were neither PKH67-positive, von Willebrand factor-positive endothelial cells nor PKH67-positive, Ki-67-positive proliferating cells in the stomach (data not shown). On day 21 (i.e., 3 wk after the transplantation), PKH67-labeled MSCs existed in the gastric submucosal and muscular layers where the cells were supposed to be injected (Fig. 7). These results suggested that most of the transplanted MSCs and their descendants survive during and after ulcer healing.


Figure 5
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 5. Light microscopic and fluorescence microscopic images of acetic acid-induced ulcers in rats treated with MSC transplantation (1x107 cells). A: epithelial regeneration was observed at the ulcer margin with dilated gastric glands. The submucosal area beneath the ulcer margin was thick, and granulation tissue was observed on day 6 [hematoxylin and eosin (H&E)]. B–D: fluorescence microscopic images showing that MSCs labeled with PKH67 were heterogeneously distributed within the submucosal layers of the gastric wall. The area marked with dotted lines in A is shown in B, C, and D: MSC staining for PKH67 (B), nuclear staining for DAPI (C), and merged (D). A: bar = 200 µm; B–D: bar = 50 µm.

 

Figure 6
View larger version (72K):
[in this window]
[in a new window]

  		Fig. 6. Differentiation of transplanted MSCs into cells of interstitial lineage in vivo. Colocalization of PKH67 (MSCs) and interstitial cell markers such as vimentin, {alpha}-smooth muscle actin ({alpha}-SMA), and desmin. MSCs labeled with PKH67 (A), vimentin labeled with rhodamine red (B), and merged (C); MSCs labeled with PKH67 (D), {alpha}-SMA labeled with rhodamine red (E), and merged (F); MSCs labeled with PKH67 (G), desmin labeled with rhodamine red (H), and merged (I) (bar = 5 µm). Specificity of rhodamine red-labeled immunohistochemistry was confirmed by using a labeled control IgG. J: percentages of transplanted MSCs expressing interstitial cell markers. Data from 4 rats are expressed as means ± SD.

 

Figure 7
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 7. Light microscopic and fluorescence microscopic images of complete reepithelialization of acetic acid-induced gastric ulcers in rats treated with MSC transplantation (1x107 cells). A: complete epithelial reepithelialization was observed on day 21 (H&E). B–D: fluorescence microscopic images showing that MSCs labeled with PKH67 were heterogeneously distributed within the submucosal layers of the gastric wall. The area marked with dotted lines in A is shown in B, C, and D. MSC-staining for PKH67 (B), nuclear staining for DAPI (C), and merged (D). A: bar = 200 µm; B–D: bar = 100 µm.

 
MSCs expressed VEGF and HGF in the gastric wall. We also explored the mechanisms underlying the accelerated healing induced by transplanted MSCs. To examine whether the transplanted MSCs secreted growth factors such as VEGF and HGF, sections of stomach were examined for colocalization of PKH67 and growth factors (VEGF and HGF). VEGF and HGF-{alpha} immunostaining were observed in PKH67-positive MSCs surrounding the ulcer, suggesting local secretion of VEGF and HGF from the transplanted MSCs (Fig. 8A). The percentages of transplanted MSCs that expressed VEGF and HGF-{alpha} were 18.6 ± 18.1 and 9.6 ± 6.8%, respectively (Fig. 8B). It is noteworthy that some MSCs expressing VEGF or HGF-{alpha} were not necessarily thin-spindle shaped. Rather, a majority of MSCs expressing VEGF or HGF-{alpha} were oval or thick-spindle shaped. VEGF and HGF-{alpha} were also expressed in PKH67-negative, spindle-shaped cells, confirming that host-derived cells of interstitial lineage might be alternative sources of VEGF and HGF in the stomach. These results suggested that MSCs accelerated gastric ulcer healing via the secretion of growth factors such as VEGF and HGF.


Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 8. Expression of VEGF and HGF in MSCs after transplantation, examined by confocal microscopy. A: colocalization of PKH67 (MSCs) and growth factors such as VEGF and HGF. MSCs labeled with PKH67 (a), VEGF labeled with rhodamine red (b), and merged (c). MSCs labeled with PKH67 (d), HGF-{alpha} labeled with rhodamine red (e), and merged (f) (bar = 5 µm). PKH67-positive MSCs were surrounded by VEGF staining and HGF-{alpha} staining. Specificity of immunohistochemistry was confirmed by using rhodamine red-labeled control IgG. DAPI staining was used for identification of nuclei. These images were omitted from the figures. B: percentages of transplanted MSCs expressing growth factors. Data from 4 rats are expressed as means ± SD.

 
VEGF immunoneutralization. Finally, we examined the involvement of VEGF in gastric ulcer healing accelerated by transplanted MSCs. Anti-VEGF neutralizing antibody dose dependently inhibited the accelerated gastric ulcer healing induced by the transplantation of the bone marrow-derived MSCs. The neutralizing antibody (at a dose of 10 µg per animal per day) significantly inhibited the gastric ulcer healing induced by the transplanted MSCs (Fig. 9). The results suggested that VEGF was involved in the accelerated gastric ulcer healing induced by MSC transplantation.


Figure 9
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 9. Effects of anti-VEGF neutralizing antibody on the size of acetic acid-induced ulcers in rats treated with MSC transplantation. Rats with acetic acid-induced gastric ulcers were killed on day 6 after the start of ulcer healing, and ulcer diameter was measured under microscopy. Data from 6–12 rats are expressed as means ± SD. *P < 0.05 vs. PBS-treated control: #P < 0.05 vs. the group treated with MSC transplantation + nonimmunized goat IgG.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that bone marrow-derived dish-adherent cells were expandable and implantable to the gastric wall and accelerated healing of acetic acid-induced ulcers, which is a model for chronic and recurring gastric ulcers (12). The cells expressed CD29 and CD90, but not CD34 and CD45, and were able to differentiate into adipocytes and osteocytes. The cells isolated from bone marrow are multipotent cells of nonhematopoietic lineages and therefore can be defined as bone marrow-derived MSCs. Previous studies (1, 3, 10, 11) reported that bone marrow-derived stem cells or progenitor cells contribute to gastrointestinal tissue restoration after a wide range of injuries. Little is known, however, about the therapeutic potential of adult somatic stem cells from bone marrow for gastrointestinal disorders.

In the present study, the bone marrow-derived MSCs transplanted into the gastric wall accelerated healing of chronic gastric ulcers. Histological and molecular characterization of the engrafted MSCs is particularly important, since the topical transplantation of MSCs can be applied to heal chronic and recurring ulcers, which are typically found in patients with Helicobacter infection, drug use, or Crohn's disease. Most experimental ulcers including those induced by physical stress or by luminal application of acetic acid are acute ulcers that heal within a few days and never recur (19). In a histopathological view, acute ulcers are shallow ulcers or erosions. On the other hand, chronic ulcers are defined as deep tissue defects penetrating beyond proper muscle layers. Serosal application or submucosal injection of acetic acid to gastric angle reproducibly induces penetrating ulcers that are slow to heal and recur spontaneously and in response to TNF-{alpha} (12). After the transplantation, most of the engrafted MSCs were positively stained for vimentin, an interstitial cell marker. These results suggest that the MSCs have phenotypes of interstitial lineage cells in the gastric wall after the local injection. Indeed, minor population of the MSCs also expressed desmin or {alpha}-SMA, suggesting that they were able to differentiate further to smooth muscle cells and myofibroblasts in gastric wall. Differentiated cells of interstitial lineage are able to produce extracellular matrixes and have important roles in wound healing via scar formation (29). We also demonstrated an important role of myofibroblasts, stimulated by gastric endothelin-1, in ulcer healing via modulating fibrosis at the lesion site (18).

The results also demonstrated that most of the engrafted MSCs and their offsprings were positive only for vimentin, and not for {alpha}-SMA or desmin. Therefore, the majority of the cells might not differentiate further to myofibroblasts or to smooth muscle cells within 9 days after the transplantation. PKH67-positive cells still existed in gastric wall that has healed from acetic acid ulcer (i.e., on day 21), indicating that the labeled MSCs and their descendants survived during the process of ulcer healing. The fate of the engrafted MSCs after the complete healing remain to be investigated in animal trials using genetically labeled MSCs such as those obtained from green-fluorescent protein-transgenic rats.

MSCs are reported to transdifferentiate into vascular endothelial cells in the rat heart (17). We were unable to observe PKH67-positive, von Willebrand factor-positive vascular endothelial cells in the rat stomach in this particular model. Transdifferentiation of the engrafted MSCs into endothelial cells appears to be an unlikely event after injection into the gastric wall.

On the other hand, the present study demonstrated that bone marrow-derived MSCs expressed mRNAs for certain angiogenic factors, VEGF and HGF, and secreted more VEGF than bone marrow cells in vitro. Because they are two potent endothelial mitogens with distinct signal transduction pathways (6), the expression of VEGF and HGF in the engrafted MSCs after transplantation was also examined in rats in vivo. Immunofluorescence microscopy clearly demonstrated that a majority of the transplanted MSCs maintained their phenotype to produce VEGF and HGF. Finally, a neutralizing antibody against VEGF dose dependently suppressed gastric ulcer healing accelerated by the transplanted MSCs. Taken together, the results suggest that local injection of MSCs promotes healing of acetic acid-induced ulcers, which is, at least in part, mediated by VEGF from the engrafted MSCs.

Since anti-HGF neutralizing antibodies were unavailable, the present study could not examine the influences of HGF on MSC-induced ulcer healing. In the present study, however, immunoneutralization of VEGF significantly delayed, but did not completely block, the gastric ulcer healing process. The results suggest the involvement of factors other than VEGF in the ulcer healing process. HGF is one of the established angiogenic factors and therefore may be involved in angiogenesis during ulcer healing. Furthermore, HGF not only is an angiogenic factor but is also a potent stimulus for gastric epithelial proliferation (27, 28, 31) and morphogenesis (31). The importance of this pleiotropic peptide in tissue regeneration in general (12, 13) and in ulcer healing (29) is well described. Therefore, the potential roles of HGF in MSC-induced acceleration of ulcer healing remain to be investigated.

In conclusion, bone marrow-derived MSCs were isolated and expanded in vitro and were transplantable into the gastric tissue in rats in vivo. The engrafted MSCs successfully accelerated gastric ulcer healing. After transplantation, minor populations of MSCs become potential sources of gastric myofibroblasts and fibroblasts, which have important roles in gastrointestinal ulcer healing via scar formation. Majority of MSCs, however, reside and survive within gastric wall for at least 21 days after the topical transplantation. The MSCs also expressed VEGF and HGF, important factors for angiogenesis and ulcer healing. The beneficial effects of the MSCs might be mediated, at least in part, by their ability to differentiate into cells of interstitial lineage and by their ability to supply large amounts of angiogenic factors. These findings may be of not only pathophysiological but also of clinical relevance, since chronic gastrointestinal ulcers can be recognized and be treated under endoscopy. Thus MSC transplantation has potential as a new therapeutic strategy for intractable gastrointestinal ulcers.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Tsuji, Dept. of Gastroenterology and Hepatology, Osaka Univ. Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan (e-mail: stsuji{at}gh.med.osaka-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Andoh A, Bamba S, Fujiyama Y, Brittan M, Wright NA. Colonic subepithelial myofibroblasts in mucosal inflammation and repair: contribution of bone marrow-derived stem cells to the gut regenerative response. J Gastroenterol 40: 1089–1099, 2005.[CrossRef][Web of Science][Medline]
  2. Askenasy N, Farkas DL. Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo. Stem Cells 20: 501–513, 2002.[Abstract/Free Full Text]
  3. Brittan M, Chance V, Elia G, Poulsom R, Alison MR, MacDonald TT, Wright NA. A regenerative role for bone marrow following experimental colitis: contribution to neovasculogenesis and myofibroblasts. Gastroenterology 128: 1984–1995, 2005.[CrossRef][Medline]
  4. Brittan M, Hunt T, Jeffery R, Poulsom R, Forbes SJ, Hodivala-Dilke K, Goldman J, Alison MR, Wright NA. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 50: 752–757, 2002.[Abstract/Free Full Text]
  5. Deb-Joardar N, Thuret G, Dumollard JM, Absi L, Campos-Guyotat L, Peoc'h M, Garraud O, Gain P. Experimental study of the survival of metastatic cancer cells in corneal organ culture. Invest Ophthalmol Vis Sci 47: 1339–1347, 2006.[Abstract/Free Full Text]
  6. Gerritsen ME, Tomlinson JE, Zlot C, Ziman M, Hwang S. Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells. Br J Pharmacol 140: 595–610, 2003.[CrossRef][Web of Science][Medline]
  7. Grogaard HK, Sigurjonsson OE, Brekke M, Klow NE, Landsverk KS, Lyberg T, Eriksen M, Egeland T, Ilebekk A. Cardiac accumulation of bone marrow mononuclear progenitor cells after intracoronary or intravenous injection in pigs subjected to acute myocardial infarction with subsequent reperfusion. Cardiovasc Revasc Med 8: 21–27, 2007.[CrossRef][Medline]
  8. Hayashi Y, Tsuji S, Tsujii M, Nishida T, Ishii S, Nakamura T, Eguchi H, Kawano S. The transdifferentiation of bone-marrow-derived cells in colonic mucosal regeneration after dextran-sulfate-sodium-induced colitis in mice. Pharmacology 80: 193–199, 2007.[CrossRef][Web of Science][Medline]
  9. Jiang S, Haider HK, Idris NM, Lu G, Ashraf M. Stable therapeutic effects of mesenchymal stem cell-based multiple gene delivery for cardiac repair. Cardiovasc Res 2008 Jan 17 [Epub ahead of print].
  10. Komori M, Tsuji S, Tsujii M, Murata H, Iijima H, Yasumaru M, Nishida T, Irie T, Kawano S, Hori M. Efficiency of bone marrow-derived cells in regeneration of the stomach after induction of ethanol-induced ulcers in rats. J Gastroenterol 40: 591–599, 2005.[CrossRef][Web of Science][Medline]
  11. Komori M, Tsuji S, Tsujii M, Murata H, Iijima H, Yasumaru M, Nishida T, Irie T, Kawano S, Hori M. Involvement of bone marrow-derived cells in healing of experimental colitis in rats. Wound Repair Regen 13: 109–118, 2005.[CrossRef][Web of Science][Medline]
  12. Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration. Biochem Biophys Res Commun 239: 639–644, 1997.[CrossRef][Web of Science][Medline]
  13. Matsumoto K, Nakamura T. Roles of HGF as a pleiotropic factor in organ regeneration. EXS 65: 225–249, 1993.[Medline]
  14. Matsumoto T, Okamoto R, Yajima T, Mori T, Okamoto S, Ikeda Y, Mukai M, Yamazaki M, Oshima S, Tsuchiya K, Nakamura T, Kanai T, Okano H, Inazawa J, Hibi T, Watanabe M. Increase of bone marrow-derived secretory lineage epithelial cells during regeneration in the human intestine. Gastroenterology 128: 1851–1867, 2005.[CrossRef][Web of Science][Medline]
  15. Melnicoff MJ, Morahan PS, Jensen BD, Breslin EW, Horan PK. In vivo labeling of resident peritoneal macrophages. J Leukoc Biol 43: 387–397, 1988.[Abstract]
  16. Mitjavila-Garcia MT, Simonin C, Peschanski M. Embryonic stem cells: meeting the needs for cell therapy. Adv Drug Delivery Res 57: 1935–1943, 2005.[Web of Science][Medline]
  17. Nagaya N, Kangawa K, Itoh T, Iwase T, Murakami S, Miyahara Y, Fujii T, Uematsu M, Ohgushi H, Yamagishi M, Tokudome T, Mori H, Miyatake K, Kitamura S. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 112: 1128–1135, 2005.[Abstract/Free Full Text]
  18. Nishida T, Tsuji S, Kimura A, Tsujii M, Ishii S, Yoshio T, Shinzaki S, Egawa S, Irie T, Yasumaru M, Iijima H, Murata H, Kawano S, Hayashi N. Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors. Am J Physiol Gastrointest Liver Physiol 290: G1041–G1050, 2006.[Abstract/Free Full Text]
  19. Okabe S, Amagase K. An overview of acetic acid ulcer models—the history and state of the art of peptic ulcer research. Biol Pharm Bull 28: 1321–1341, 2005.[CrossRef][Web of Science][Medline]
  20. Okamoto R, Yajima T, Yamazaki M, Kanai T, Mukai M, Okamoto S, Ikeda Y, Hibi T, Inazawa J, Watanabe M. Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal tract. Nat Med 8: 1011–1017, 2002.[CrossRef][Web of Science][Medline]
  21. Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103: 1662–1668, 2004.[Abstract/Free Full Text]
  22. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147, 1999.[Abstract/Free Full Text]
  23. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276: 71–74, 1997.[Abstract/Free Full Text]
  24. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337–346, 2002.[CrossRef][Web of Science][Medline]
  25. Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, Sato T, Miyanishi K, Takayama T, Takahashi M, Takimoto R, Iyama S, Matsunaga T, Ohtani S, Matsuura A, Hamada H, Niitsu Y. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 106: 756–763, 2005.[Abstract/Free Full Text]
  26. Suzuki N, Takahashi S, Okabe S. Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats. J Physiol Pharmacol 49: 515–527, 1998.[Web of Science][Medline]
  27. Takahashi M, Ota S, Shimada T, Hamada E, Kawabe T, Okudaira T, Matsumura M, Kaneko N, Terano A, Nakamura T. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture. J Clin Invest 95: 1994–2003, 1995.[Web of Science][Medline]
  28. Takahashi M, Ota S, Terano A, Yoshiura K, Matsumura M, Niwa Y, Kawabe T, Nakamura T, Omata M. Hepatocyte growth factor induces mitogenic reaction to the rabbit gastric epithelial cells in primary culture. Biochem Biophys Res Commun 191: 528–534, 1993.[CrossRef][Web of Science][Medline]
  29. Tarnawski AS. Cellular and molecular mechanisms of gastrointestinal ulcer healing. Dig Dis Sci 50, Suppl 1: S24–S33, 2005.[CrossRef][Web of Science][Medline]
  30. Teare GF, Horan PK, Slezak SE, Smith C, Hay JB. Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes. Cell Immunol 134: 157–170, 1991.[CrossRef][Web of Science][Medline]
  31. Tsuji S, Kawano S, Tsujii M, Fusamoto H, Kamada T. Roles of hepatocyte growth factor and its receptor in gastric mucosa. A cell biological and molecular biological study. Dig Dis Sci 40: 1132–1139, 1995.[CrossRef][Web of Science][Medline]
  32. Zhao FQ, Zhang PX, He XJ, Du C, Fu ZG, Zhang DY, Jiang BG. Study on the adoption of Schwann cell phenotype by bone marrow stromal cells in vitro and in vivo. Biomed Environ Sci 18: 326–333, 2005.[Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/G778    most recent
00468.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, Y.
Right arrow Articles by Kawano, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, Y.
Right arrow Articles by Kawano, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.