Gastroenterology
Volume 132, Issue 3 , Pages 944-954, March 2007

Nonmyeloablative Stem Cell Therapy Enhances Microcirculation and Tissue Regeneration in Murine Inflammatory Bowel Disease

  • Philipe N. Khalil

      Affiliations

    • Department of Surgery, Klinikum Innenstadt, Ludwig-Maximilians-Universität of München, Munich, Germany
    • Corresponding Author InformationAddress requests for reprints to: Philipe N. Khalil, MD, Department of Surgery, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, Nuβbaumstraβe 20, 80336 Munich, Germany. fax: (49) 89-5160-4722.
    • ,
  • Véronique Weiler

      Affiliations

    • Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany
    • ,
  • Peter J. Nelson

      Affiliations

    • Department of Clinical Biochemistry, Medizinische Poliklinik, Ludwig-Maximilians-Universität München, Munich, Germany
    • ,
  • Maurice N. Khalil

      Affiliations

    • Department of Surgery, Klinikum Innenstadt, Ludwig-Maximilians-Universität of München, Munich, Germany
    • ,
  • Sabine Moosmann

      Affiliations

    • Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany
    • ,
  • Wolf E. Mutschler

      Affiliations

    • Department of Surgery, Klinikum Innenstadt, Ludwig-Maximilians-Universität of München, Munich, Germany
    • ,
  • Matthias Siebeck

      Affiliations

    • Department of Surgery, Klinikum Innenstadt, Ludwig-Maximilians-Universität of München, Munich, Germany
    • ,
  • Ralf Huss

      Affiliations

    • Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany

Received 17 July 2006; accepted 16 November 2006. published online 30 December 2006.

Article Outline

Background & Aims: Reduced microcirculation has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Stem cells or endothelial progenitor cells are thought to contribute to tissue regeneration through neoangiogenesis or vasculogenesis in ischemia- or inflammatory-related diseases. We therefore hypothesized that adult stem cells facilitate epithelial repair in IBD. Methods: Moderate–severe colitis in mice was induced by dextran sulfate sodium (DSS) and 2.0 × 106 immortalized CD34− stem cells infused twice via the tail vein during an observation period of 35 days in a nonmyeloablative setting. Results: Here, we demonstrate that adult stem cells home to the damaged digestive tract in the large intestine and facilitate mucosal repair in moderate-severe colitis. Nonmyeloablative stem cell therapy resulted in increased survival in severe colitis (P < .0001). Moreover, clinical activity and histologic evaluation of the colitis severity score were reduced significantly in moderate (P = .0003 or P = .03) and severe (P < .0001 or P < .03) colitis after 35 days, in addition to the DSS-induced shortening of colon length (P = .002 and P < .0002). Genetically marked stem cells were detected predominately in the submucosa of the damaged colon epithelium. Epithelial repair in experimental IBD was mediated either by induction of improved vasculogenesis or by the differentiation of the transplanted stem cells into endothelial cells, as demonstrated by the promotion of Tie2 activity in the infused cells at the site of the damaged mucosa. Conclusions: Our findings indicate that systemically administered adult stem cells respond to an adequate tissue lesion in murine IBD by enhancing microcirculation, resulting in accelerated tissue repair.

Abbreviations used in this paper: CAS, clinical activity score, DAPI, 4’,6’-diamidino-2-phenylindole, DSS, dextran sulfate sodium, Flt-1, FMS-like tyrosine kinase 1 or vascular endothelial growth factor receptor (VEGF-R1), GFP, green fluorescence protein, H&E, hematoxylin & eosin, MSC, mesenchymal stem cells, MW, molecular weight, PECAM-1, platelet-endothelial cell adhesion molecule (CD31 antigen), RFP, red fluorescence protein, SC, stem cells, SCL, stem cell leukemia or stem-cell leukemia hematopoietic transcription factor (tal1), SV40, simian virus 40, Tie2, endothelium-specific receptor tyrosine kinase 2

 

See editorial on page 1171.

Inflammatory bowel disease (IBD), comprising Crohn’s disease and ulcerative colitis, is thought to result from inappropriate and ongoing activation of the mucosal immune response driven by the presence of the normal intestinal microflora. This aberrant response is most likely facilitated by defects of the epithelial barrier function and the mucosal immune system, leading to long-term and sometimes irreversible impairment of gastrointestinal structure and function.1, 2 The histopathologic features of Crohn’s disease resemble those of experimental T-helper 1-cell-mediated colitis, whereas those of ulcerative colitis are most similar to experimental TH2-mediated colitis.1, 3, 4, 5, 6 The current standard drug regimen for IBD is focused on suppression and control of inflammation using 5-aminosalicylates, corticosteroids, and immune-modulating drugs such as azathioprine and mercaptopurine.7 Despite advances in understanding the pathophysiology and some progress in the management of IBD, no new therapeutic strategies have been developed in recent years.8, 9 Nevertheless, despite ablation of the possible pathogenic cause and control of the ongoing immune process, the epithelial damage and its immunologic barrier need to be repaired.

Emerging evidence suggests that adult stem cells (eg, from the bone marrow) contribute to tissue regeneration partly by promoting neovascularization or arteriogenesis. This may occur through the release of chemokines that recruit progenitor cells and circulating endothelial progenitor cells to the site of the lesion and modify the lineage commitment needed for new vessel growth.10, 11, 12 Tissue ischemia usually leads to up-regulation of angiogenic factors, promoting migration of the progenitor cells to the site of the lesion.10, 11, 13, 14, 15 Indeed, there is a growing list of disorders characterized or caused by abnormal or inadequate angiogenesis. An abnormal microcirculatory system has been implicated in the pathogenesis of IBD that may contribute to reduced mucosal perfusion, poor wound healing, and maintenance of chronic inflammation.15, 16, 17, 18 Based on these reports, we hypothesized that circulating and/or therapeutically transfused adult stem cells would recognize the IBD lesion, home to the damaged digestive tract, and facilitate epithelial repair by promoting angiogenesis. Thus, experimental colitis in mice was induced by dextran sulfate sodium (DSS), which leads to epithelial damage and thereby stimulates a local inflammatory response through the production of cytokines and other inflammatory mediators via the TH2-like pathway.1, 3, 4, 19 We have previously shown that CD34− adult stem cells can achieve complete lymphohematopoietic reconstitution after myeloablative therapy, as well as improved angiogenesis with simultaneous skeletal muscle repair, in a murine ischemia model.20, 21 Here, we examine whether therapeutic nonmyeloablative stem cell treatment could facilitate epithelial repair and thereby prevent the progressive weight loss and bloody diarrhea that occurs in acute murine colitis. Cultured adult CD34− stem cells were administered twice via tail vein injection. Clinical, gross morphologic and histomorphologic scores were used to assess both the therapeutic effects of stem cells and the homing and differentiation of transplanted and genetically marked stem cells to estimate the degree of mucosal repair and vascularization in the affected colonic regions.

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Materials and Methods 

Animals 

Eighty-four female 6- to 8-week-old BALB/c mice, weighing 20–23 g, were obtained from the Institute of Pathology (Ludwig-Maximilians-University, Munich, Germany) and housed in groups of 3 animals in an open environmental facility using metabolic cages (Reference number 3700M022, Techniplast Gazzada S.ar.l., Buguggiate, Italy). The mice were kept under a 12-hour day/night cycle at 20°C–22°C and had free access to food and water during the study. Mice were killed by cervical dislocation under ether anesthesia. The protocol used was approved by the Animal Care and Use Committee at the Government of Upper Bavaria in Munich and was in direct accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Murine Colitis Model 

Acute colitis in mice was induced by DSS administered orally in a cyclic manner, which caused reproducible histologic inflammation mainly in the left colon.4, 19 Briefly, mice received 2 cycles of treatment with DSS (MW 40,000 daltons; MP Biomedicals, Brussels, Belgium) under a prospective randomized study protocol. Each cycle consisted of 7 days with either 3% (n = 30) or 5% (n = 36) DSS added to the drinking water, followed by a 10-day period without DSS supplementation to generate a spectrum from moderate to severe intestinal inflammation. Two further groups of mice receiving no DSS (each n = 9) served as controls. On day 8 of each cycle, mice were injected with 2.0 × 106 CD34− stem cells in the tail vein.

Murine Adult Stem Cell Clones 

Murine adult CD34− stem cell lines were established from the bone marrow and peripheral blood of BALB/c and CBA mice as described previously.20, 21 Clonality of the cell lines was achieved by immortalization of the primary cells with SV40 large-T antigen, followed by cellular cloning and subcloning. Stem cells were cultured under standard conditions until 80% cell confluency was reached for further use or evaluation. Flow cytometry and transcriptional analysis revealed a strong angiogenic potential of the established cell lines.21 Stem cells were also stably transfected with green fluorescence protein (GFP) for the detection of transplanted cells after integration into tissues or with a pTie2-RFP construct (endothelial cell-specific promoter-driven red florescence protein). Stable integration of the constructs was selected for using G418 or blastacin. Transplanted cells were visualized in colon tissue with an antibody against SV40 large-T antigen after formalin fixation and paraffin embedding (Oncogene Research Products, Boston, MA) using an indirect immunoperoxidase method (Picture PLUS Kit; Zymed, San Francisco, CA).21 Endogenous peroxidase activity was blocked by 3% H2O2. Optimal labelling of SV40 staining in the transplanted cells in situ was obtained after microwave pretreatment of semithin paraffin sections (2 cycles of 15 minutes in Target Retrieval Solution; Dako, Hamburg, Germany). SV40 peroxidase activity was finally detected by addition of the Vector VIP substrate kit (Vector Laboratories, Burlingame, CA). For fluorescence staining, slides were also counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma, Deisenhofen, Germany) to detect the chromatin structure of the nuclei.

Transfusion of Stem Cells Into Host Animals 

Adult stem cells from the cell lines were isolated from the culture flasks by trypsin digestion for 5 minutes at 37°C and thoroughly washed in 0.9% NaCl. After washing, 2.0 × 106 stably transfected cells (GFP or pTie2-RFP) in a volume of 200 μL per mouse were injected directly into a tail vein without ablation as previously described.22 The distribution of cells was assessed by the detection of green or red fluorescence in the tissue environment with or without DSS pretreatment. Differentiation of stem cells into endothelial cells was assessed by the expression of RFP after activation of the endothelial cell-specific Tie2 promoter.

Clinical Assessment of the Severity of Colitis 

The clinical activity score (CAS) was assessed by body weight, stool consistency, and occult blood (measured by the guaiac reaction, hemoccult) according to Okayasu et al.19 No weight loss was scored as 0 points, weight loss of 1%–5% as a score of 1, 5%–10% as 2, 10%–20% as 3, and >20% as 4. For stool consistency, 0 points were given for well-formed pellets, 2 for pasty and semiformed stools that did not stick to the anus, and 4 for watery diarrhea that did stick to the anus. Bleeding was scored as 0 for the hemoccult-negative test, 2 for hemoccult positive, and 4 for gross bleeding. The individual scores were added and divided by 3, resulting in a CAS score ranging from 0 to 4.

Macroscopic and Histomorphologic Assessment of the Severity of Murine Colitis 

Postmortem, the entire colon was removed from the cecum to the anus, flushed with saline, and placed without tension on cellulose. Colon length and weight were measured as indirect markers of inflammation.4, 19, 20 The entire colon was fixed in 4% paraformaldehyde overnight before paraffin embedding. Serial 1.0- to 2.0-μm-thick sections were sliced from the paraffin block, and the slides were deparaffinized in xylene and dried in ethanol before staining with H&E for histologic scoring by 2 pathologists blinded to the identity of the samples. The macroscopic colonic damage score was assessed following Wallace et al,23 with modification, during removal of the entire colon, and comprised the grade of tissue adhesion, presence of ulceration, and wall thickness.23 No adhesions were scored as 0, little, or moderate effort required to separate the colon from the surrounding tissue as 1 or 2, and severe adhesions as 3. Normal appearance of the colon and focal hyperemia with no ulcers were scored as 0 and 1, respectively, the presence of ulcers and inflammation was scored 2, and 2 or more ulcers and regions of inflammation as 3. A wall that appeared to be of normal thickness was scored as 0, mild or moderate thickening as 1 or 2, and severe bowel thickening as 3. The individual scores for macroscopic colon damage were added, resulting in a range from 0 to 9. A combined score was used next to assess the histologic degree and extent of inflammation and tissue damage.24, 25 To assess the infiltration of inflammatory cells, rare inflammatory cells in the lamina propria were scored as 0, increased numbers of inflammatory cells in the lamina propria as 1, confluence of inflammatory cells extending into the submucosa as 2, and transmucosal infiltrates as 3. For determination of tissue damage, no mucosal damage was scored as 0, a discrete lymphoepithelial lession as 1, surface mucosal erosion as 2, and extensive mucosal damage and ulceration as 3. The scores for cell infiltration and tissue damage were added, resulting in a combined histologic score ranging from 0 to 6.

Statistics 

Analyses and preparation of the figures were performed using the Statistical Package for the Social Sciences (version 10.1; SPSS Inc, Chicago, IL) and Software for Scientific Graphing (version 6.1; Origin Lab Corporation, Northampton, MA). Results were expressed as mean ± standard error of mean (SEM); n refers to the quantity of animals. Kaplan-Meier survival curves were compared using the log-rank test. Means between groups were compared with the unpaired Student t test, and, where necessary, analysis of variance (ANOVA) was used for multiple comparisons. A 2-sided test at an α level of <.05 rejecting alternative hypotheses was considered statistically significant.

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Results 

CD34− Stem Cells Reduce Clinical Activity in Murine Colitis 

In BALB/c mice, 2-cycle feeding of 3% DSS for 7 days and a 10-day recovery period without DSS added to the drinking water resulted in only moderate acute colitis, whereas 5% DSS produced severe colitis with a mortality of 81%, compared with 40% in the 3% DSS group, during the observation period of 35 days. All mice receiving 3% or 5% DSS additives developed constant clinical signs of colitis within 2–4 days, including weight loss and bloody diarrhea. CD34− stem cells were transfused twice after the induction of acute colitis, on day 8 and day 25. Control mice receiving no DSS in the drinking water did not develop any signs of colitis and gained weight over time with or without stem cell therapy. Both cycles of 7 days of addition of DSS to the drinking water resulted in a significant reduction in body weight and occurrence of diarrhea and bloody stools, which were combined in the CAS. The course of intestinal inflammation was characterized by a biphasic cycle of increasing and decreasing clinical activity induced by DSS (Figures 1A and 1B). Thus, mice recovered partly during both 10-day resting periods without DSS, and a decrease in the CAS was therefore observed. In the 3% and 5% DSS groups, no significant differences in CAS were seen after the first cycle of 7 days of DSS, on day 8 before stem cell administration (DSS 3%, 2.4 ± 0.2 vs 2.4 ± 0.3, P = .9; DSS 5%, 3.1 ± 0.7 vs 3.3 ± 0.6, P = .4), indicating comparability within the DSS concentration groups. However, mean CAS improved significantly after the first and second stem cell transfusions in both DSS concentration groups, whereas the CAS remained significantly elevated in the corresponding DSS control groups after 35 days (DSS 3%, 1.8 ± 0.4; DSS 5%, 1.9 ± 0.1). CASs were similar after 35 days and 2 stem cell transfusions in the stem cell groups (DSS 3%, 0.3 ± 0.1; DSS 5%, 0.4 ± 0.1), resulting in significant differences between groups (DSS 3% vs DSS 3% + stem cell [SC], P < .0003; DSS 5% vs DSS 5% + SC, P < .00002) (Figure 1A and 1B). A greater decrease in the CAS, toward fewer clinical signs of inflammation, was noted in the 5% DSS compared with the 3% DSS group after the first stem cell transfusion; this was most likely related to the higher mortality and therefore drop out of the most severely ill 5% DSS mice. No notable clinical activity was observed in control mice, regardless of whether they received CD34− stem cell therapy in addition to normal tap water (Figure 1C). We therefore presume that the experimental protocol had no influence on the CAS. The individual scores of body weight, rectal bleeding, and stool consistency in the 5% and 3% DSS feeding groups after 35 days are shown separately in Figures 1D and 1E. Two CD34− stem cell infusions resulted in markedly increased survival in moderate and severe murine colitis (Figures 1F and 1G). However, only in the 5% DSS group did the increased survival reach statistical significance (P = .0001 compared with P = .18; Figure 1G). CD34− stem cells reduced mortality by 50% and 60% in the 3% and 5% DSS groups, respectively. None of the control mice, regardless of whether they received stem cell therapy, died prematurely.

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  • Figure 1. 

    Effect of adult CD34− stem cell therapy on the clinical activity in severe and moderate murine colitis. BALB/c mice received either 5% or 3% dextran sulfate sodium (DSS) with the drinking water in cyclic manner. Each cycle consisted of 7 days DSS followed by a 10-day period without DSS supplementation. Immortalized adult CD34− stem cells (SC) (2.0 × 106) were infused via the tail vein on days 8 and 25 without previous whole body ablation of the hosts. An additional 2 groups receiving no DSS but tap water throughout served as controls. Additionally, mice in one of the control groups received stem cells by tail vein injection as described. Mice receiving 3% (A) or 5% (B) DSS twice showed an increasing and decreasing clinical activity in contrast to controls (C) during the observation period of 35 days as obvious from the clinical activity score (CAS). The individual score, consisting of weight loss, stool consistency, and rectal bleeding is broken down separately at day 35 for the 3% and 5% DSS groups (D and E). Figures F and G show the Kaplan-Meier survival plots in mice fed with either 3% (F) or 5% (G) DSS. Thus, stem cell therapy reduced mortality by 50% and 60% in the 3% and 5% DSS feeding groups, respectively. Higher survival was statistically significant in severe colitis (P = .0001) but not in moderate colitis (P = .18). *P < .05, †P < .01, ‡P < .001, #P < .0001 for comparison between groups.

Evidence of CD34− Stem Cell Homing and Promotion of Microcirculation in Murine Colitis 

Transfused stem cells were stably transfected with GFP to detect possible stem cell homing to the inflamed colon. The fluorescein signal was exclusively detected in the mucosa and submucosa of the inflamed colon but not in unaffected colonic segments or in control mice. Immunohistochemical staining of the SV40 large-T antigen of the stem cells showed these cells in the close vicinity of sprouting vessels (Figure 2A–C). To investigate whether stem cells would promote neovascularization, we transfected the stem cells with a pTie2-RFP construct to visualize potential endothelial differentiation of the stem cells.26 Here, adult stem cells were found to differentiate into Tie2-expressing cells that corresponded with an improvement in mucosal perfusion and overall microcirculation in the damaged tissue (Figure 2D–F).

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  • Figure 2. 

    Detection and differentiation of transfused adult CD34− stem cells in host mice. Representative colonic slices after therapeutic stem cell therapy without previous whole body ablation of the hosts in mice 35 days after induction of colitis with dextran sulfate sodium (DSS). Detection of GFP-marked CD 34− stem cells (A) and stem cells with SV40 large-T antigen expression (B and C) were detected in the subepithelial stroma layer and the close vicinity of newly formed blood vessels. The transfused cells also differentiated into endothelial cells of the growing vasculature by showing an activation of the Tie2-promotor, driving the red fluorescence protein (RFP) (E). Figure D shows a DAPI staining of the colon mucosa and submucosa; (F) overlay of pictures in C and E.

CD34− Stem Cells Reduce Morphologic Signs in Murine Colitis 

We next investigated the influence of nonmyeloablative stem cell therapy with regard to morphologic inflammatory changes. Mice were killed after 35 days, and the macroscopic colonic damage score was assessed during removal of the entire colon. Stem cells significantly reduced the mean grade of macroscopic damage in both the 3% and the 5% DSS groups, (DSS 3%, 4.6 ± 0.6 vs DSS 3% + SC, 1.9 ± 0.5, P < .00005; DSS 5%, 7.8 ± 0.3 vs DSS 5% + SC, 3.4 ± 0.4, P < .005), whereas controls showed no differences (H2O, 0.08 ± 0.1 vs SC, 0.2 ± 0.2, P = .7) (Figure 3A). The entire colon was then placed on cellulose without tension for measurement of length. DSS addition led to a reduced mean colon length compared with both control groups, which did not differ from each other, as expected (DSS 3%, 10.6 ± 0.5 cm and DSS 5%, 9.1 ± 0.4 cm vs H2O, 13.4 ± 0.1 cm and SC, 13.6 ± 0.2 cm). However, the 2 CD34− stem cell transfusions significantly reduced the extent of DSS-induced colon shortening in both the 3% and the 5% DSS groups (DSS 3%, 10.6 ± 0.5 cm vs DSS 3% + SC, 12.8 ± 0.7 cm, P = .0002; DSS 5%, 9.1 ± 0.4 cm vs DSS 5% + SC, 11.5 ± 0.4 cm, P = .002) (Figure 3B). This observation of reduced colon shortening in the stem cell therapy groups was in direct accordance with our observation of a significantly lower organ weight found in the stem cell transfusion groups compared with their corresponding 3% and 5% DSS groups (DSS 3%, 0.8 ± 0.03 g vs DSS 3% + SC, 0.67 ± 0.02 g, P < .007; DSS 5%, 0.87 ± 0.04 g vs DSS 5% + SC, 0.7 ± 0.02 g, P < .002). No difference in mean colon weight was found between control mice, independent of stem cell infusion therapy in addition to normal tap water (H2O, 0.63 ± 0.03 g vs SC, 0.68 ± 0.03 g, P > .3) (Figure 3C). Spleen weight was measured as an additional macroscopic indicator of inflammation. No difference in spleen weight was found between control groups (H2O, 0.084 ± 0.004 g vs SC, 0.109 ± 0.013 g, P = .09), whereas mean organ weight was significantly higher in the 3% and 5% DSS groups compared with the corresponding stem cell groups (DSS 3%, 0.118 ± 0.01 g vs DSS 3% + SC, 0.095 ± 0.004 g, P < .02; DSS 5%, 0.189 ± 0.269 g vs DSS 5% + SC, 0.134 ± 0.008 g, P < .02).

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  • Figure 3. 

    Effect of adult CD34− stem cell therapy on morphologic alterations in severe and moderate murine colitis. BALB/c mice received either 5% or 3% dextran sulfate sodium (DSS) with the drinking water in a cyclic manner. Each cycle consisted of 7 days DSS followed by a 10-day period without DSS supplementation. Immortalized adult CD34− stem cells (SC) (2.0 × 106) were infused via the tail vein on days 8 and 25 without previous whole body ablation of the hosts. An additional 2 groups receiving no DSS but tap water throughout served as controls. Additionally, mice in one of the control groups received stem cells by tail vein injection as described. Mice fed with either 5% or 3% DSS and receiving stem cell therapy showed a significant lower macroscopic colonic damage score comprising grade of adhesions during removal of the entire colon, existence of ulcerations, and wall thickness compared with the corresponding controls (A). Stem cell therapy also reduced the shortening of the colon length (B) and increasing weight (C). A representative preparation of the entire alimentary system of a non-DSS-fed mouse is given in D, whereas E shows an excised colon of a mouse fed with 5% DSS and (F) a colon of a mouse fed with 5% DSS after stem cell therapy on days 8 and 25 after 35 days. The differences of colon length and wall thickness are obvious. Note the scale on the right side (D–F) and the different magnifications used. *P < .05, †P < .01, ‡P < .001, #P < .0001 for comparison between groups. NS, not significant.

CD34− Stem Cells Reduce Histologic Grade in Murine Colitis 

The histologic colonic severity scores combined the degree of inflammation and tissue damage. Consistent with our hypothesis, we found significantly reduced histologic colonic severity scores in the stem cell therapy groups compared with the corresponding DSS feeding groups (DSS 3%, 4.1 ± 0.5 vs DSS 3% + SC, 2.5 ± 0.5, P = .03; DSS 5%, 3.8 ± 0.3 vs DSS 5% + SC, 1.9 ± 0.4, P = .03) (Figure 4A). No differences were observed in severity scores between the 3% and 5% DSS groups without therapeutic CD34− stem cell infusion, most likely because of the high mortality in the 5% DSS group and the lethality above a score of 4 points (P = .7). A small ground noise of inflammation was also detected in both control groups, regardless of whether they received stem cells in addition to normal tap water, but without differences between groups (H2O, 1.0 ± 0.3 vs SC, 0.7 ± 0.2, P = .4).

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  • Figure 4. 

    Histologic characteristics of adult CD34− stem cell therapy in murine colitis. Representative colonic slices after therapeutic stem cell therapy without previous whole body ablation of the hosts in mice 35 days after induction of colitis with dextran sulfate sodium (DSS). H&E-stained paraffin sections of murine colon mucosa and submucosa after 2 cycles of feeding 5% DSS with the drinking water and without CD34− stem cell therapy (A and B) show typical deep ulcerative damage; disordered mucosal architecture; diffuse depletion of goblet cells; and increased number of inflammatory cells (mononuclear cells and granulocytes) within the epithelium, the lamina propria, and the submucosa (original magnification in A, ×200; B, ×400). Mice that underwent stem cell therapy via the tail vein without previous whole body ablation for 5% DSS-induced colitis (C and D) showed only discrete epithelial lesion of the epithelium, focal depletion of goblet cells, and less inflammatory cells (mostly mononuclear cells) within the lamina propria and the submucosa (original magnification in C, ×200; D, ×400). Regular colon mucosa receiving neither DSS nor stem cell therapy is shown in E and F. The Figure shows no lesion of the epithelium, normal mucosal architecture, regular goblet cells, and an ordinary rate of inflammatory cells within the lamina propria.

Samples from 4 different regions of the colon were used for the histologic evaluation. Mice receiving intravenous stem cell treatment showed significantly reduced areas of epithelial erosion and inflammation and fewer affected colon segments compared with the corresponding 3% and 5% DSS feeding groups (DSS 3%, 3.0 ± 0.4 vs DSS 3% + SC, 2.1 ± 0.2, P < .03; DSS 5%, 3.8 ± 0.3 vs DSS 5% + SC, 2.2 ± 0.3, P < .03). Even in the control groups, a mean of 1.0 affected colon region was observed (H2O, 1.0 ± 0.2 vs SC, 1.0 ± 0.4, P = 1.0), most likely related to the open environmental housing and feeding conditions in the animal facility (Figure 4B). It became obvious from representative H&E-stained colon tissue that CD34− stem cell treatment reduced the degree of ulceration and mucosal damage in severe acute colitis (Figure 5).

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  • Figure 5. 

    Effect of adult CD34− stem cell therapy on the histologic colonic severety in murine colitis. BALB/c mice received either 5% or 3% dextran sulfate sodium (DSS) with the drinking water in a cyclic manner. Each cycle consisted of 7 days followed by a 10-day period without DSS supplementation. Immortalized adult CD34− stem cells (SC) (2.0 × 106) were infused via the tail vein on days 8 and 25 without previous whole body ablation of the hosts. An additional 2 groups receiving no DSS but tap water throughout served as controls. Additionally, mice in one of the control groups received stem cells by tail vein injection as described. Mice fed with either 5% or 3% DSS and receiving stem cell therapy showed a significant lower histologic colonic severity score comprising the grade of inflammatory infiltration and tissue damage compared with controls (A). Stem cell therapy also reduced the number of affected colonic segments as detailed in B. *P < .05 for comparison between groups. NS, not significant.

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Discussion 

In the present study, the potential therapeutic role of nonmyeloablative adult stem cell therapy in experimental murine colitis was evaluated. The results suggest that adult CD34− stem cells are effective in reducing both the clinical and the pathologic features associated with IBD. A central finding of the study was the observation of clearly improved survival after therapeutic nonmyeloablative stem cell infusion that was in direct accordance with a significantly lower CAS as well as reduced shortening of the colon. Furthermore, the clinical efficacy of adult stem cell therapy in severe and moderate IBD was supported by macroscopic and histomorphologic analysis of the colon. It became obvious that regeneration of the colonic mucosa was much more efficient and accelerated in the presence of intravenously applied immortalized syngeneic stem cells.

Over the past decade, many groups have demonstrated that spindle-shaped CD34− stem cells (MSC) have the potential to differentiate into mesenchymal tissues, suggesting their multilineage differentiation capacity but not the omnipotency exhibited by embryonic stem cells.26 MSC expanded in vitro represent heterogeneous populations that include multiple generations of mesenchymal cell progeny. These populations may have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and nonmesenchymal lineages. In addition, it can be very difficult to generate sufficient numbers of cells for selective engineering using viral or nonviral vectors. To help address these various issues, syngeneic and allogeneic clones from different mouse strains were generated and evaluated for use in various models of tissue regeneration. Strikingly, whether the cell lines were immortalized using SV40 large-T antigen20, 27 or by isolating cells from P53−/− mice (Conrad et al, manuscript in preparation), they appear to have retained significant pluropotency. For example, the SV40 large-T-modified cells have been shown to reconstitute hematopoietic lineages in lethally irradiated mice.20, 27 In addition, the cells are capable of regenerating mesenchymal tissue (Huss R, manuscript in preparation). The use of the lines has also allowed serial experiments to be performed in vitro and in vivo under standardized conditions. We recently demonstrated correspondence between primary and immortalized MSC on the basis of functional chemokine receptor expression.28 In the same study, MSC clones were shown to home to various tissue environments in normal mice, including the mucosa of the small intestine, secondary lymphatic tissues, skin and salivary glands, suggesting the involvement of chemokines in the homeostasis and tissue-specific recruitment of early adult progenitor cells.28

The questions could be raised whether or not CD34− stem cells are actually the cell population needed and whether the same number of peripheral blood cells would have the same effect. It has been shown historically that peripheral blood-derived stem cells are able to achieve similar effects as compared with bone marrow-derived stem cells.29, 30 However, their frequency is low, making it necessary to significantly enrich them or to “transplant” 50- to 100-fold more mononuclear cells from the peripheral blood as compared with bone marrow. It has been shown that CD34− stem cells can give rise to CD34+ cells, which obtain this phenotype while circulating in the peripheral blood.31 However, the state of CD34 positivity is reversible as the cells initiate mesenchymal differentiation or become quiescent.31 Therefore, the regeneration effects described here may be possible with either CD34− cells or CD34+ cells.

The experiments performed here used syngeneic cells. That is, the cells were genetically identical but derived from a different mouse. The use of syngeneic cells is analogous to the application of autologous stem cells collected from a patient and infused back to the individual. It is unclear at this time whether the similar results could be also be obtained using allogenic stem cells—disparate in at least part of the major histocompatibility complex. The potential application of allogenic cells would greatly expand the general application of this procedure as a therapeutic option.

In our experiments, the infused CD34− stem cells were detectable in the mucosa and submucosa and showed differentiation into endothelial cells, as demonstrated by the activation of the Tie2 promotor. In addition, GFP+/SV40+ cells were found in the close vicinity of newly formed blood vessels, where stem cells support the proliferation and differentiation of local endothelial precursor cells, as shown previously.21 Taking these findings together, we demonstrated for the first time direct evidence of the regenerative capacity of nonmyeloablative adult CD34− stem cell therapy in murine IBD, which responded to the epithelial damage by augmentation of neovascularization and improved microcirculation leading to accelerated tissue repair. Other authors have shown that bone marrow-derived cells are involved in the healing process in experimental colitis using different models.32, 33, 34 Komori et al detected GFP-positive bone marrow-derived cells from transgenic rats in the mucosal tissue of lethally irradiated host animals with 2,4,6-trinitrobenzenesulfonic acid-induced colitis and postulated their involvement in regeneration of the inflamed colon.34 Brittan et al used male bone marrow cells transplanted into female host BALB/c mice in a similar setting and observed both a significant contribution of the bone marrow cells to intestinal myofibroblast formation in inflamed compared with noninflamed colonic regions and the involvement of bone marrow cells in the repair and formation of blood vessels in colitis, forming endothelial cells, vascular smooth muscle cells, and pericytes.33 More recently, the same group used interleukin-10−/− mice, developing a spontaneous form of colitis during early life, as a model of IBD to investigate the involvement of bone marrow-derived cells in the inflamed mucosa. Mice receiving interleukin-10−/−-derived bone marrow cells after whole body irradiation exhibited progression of colitis, in contrast to interleukin-10−/− mice receiving wild-type-derived bone marrow cells, in which inflammation decreased.32 However, the concept of resetting the immune system by autologous stem cell transplantation following whole body myeloablation, for example in refractory Crohn’s disease, has been controversial.35, 36, 37 We, here, present the concept that stem cell treatment for IBD without previous myeloablation of the host is possible by using immortalized and well-established stem cell lines.21

There are different animal models available to investigate early events and interactions among different components and to identify the immunologic processes and genes that determine susceptibility, leading to a better understanding of this complex disorder of unknown etiology.4 Various animal models of IBD differ in their relative use, although there is no right or wrong model of IBD.4 However, DSS-induced colitis in mice is widely used and has been recommended for the investigation of new therapeutic strategies because it is easy to induce and produces a consistent degree of colitis with a defined onset.5, 38, 39

Similar to the results obtained in adult human stem cells, adult murine stem cells also express a variety of mesodermal and erythroid/myeloid transcription factors, including SCL.21, 40 Expression of endothelial markers such as PECAM-1 and Flt-1 (VEGF-R1) is particularly high when growth conditions are favorable in vitro but also in vivo. Given the ease of endothelial differentiation in vitro, the expression of endothelial marker genes and the up-regulation of angiogenic genes in actively growing cells, murine CD34− stem cells may indeed be thought of as endothelial precursor cells or hemangioblasts. The functional angiogenic properties of murine stem cell clones has been proved in an in vivo model of chronic hypoxia.21 Murine adult stem cells may be angioblast-type cells and therefore a common progenitor of hematopoietic and endothelial cells. Vasculogenesis has been shown to be a potent form of adaptive blood vessel growth in adult organisms to recover sufficient blood flow after the occlusion of a major artery.41 It has been shown that the recruitment of circulating blood cells, namely monocytes, contributes to the enlargement of preexisting collateral anastomoses to true arteries by the augmentation of cells integrated in the vessel wall.42 Here, we have shown that therapeutically infused stem cells start to differentiate into endothelial cells to improve mucosal perfusion and overall microcirculation. It was also suggested that collateral artery growth may not necessarily require the integration of circulating stem cells or endothelial progenitor cells in the growing blood vessel wall but may rather be accomplished through proliferation of the cells present in preexisting collateral vessels. Stem cells may support neovascularization by releasing stimulating and activating cytokines and growth factors, which in turn participate in the creation of an inflammatory environment that is necessary for the enhancement of arteriogenesis. It was further suggested that quiescent stem cells also reside as nonproliferating perivascular pericytes or supporting cells adjacent to the growing vasculature, releasing growth factors and stimulating the differentiation and possibly proliferation of preexisting angiogenic progenitors.43, 44 Growth factors may also recruit preexisting and primed stem cells from other reservoirs, such as the bone marrow, or directly stimulate arteriogenesis.44, 45, 46 It was also recently reported that bone marrow-derived stem cells provide functional hemangioblast activity in vivo or regulate arteriogenesis at the site of the lesion.46, 47 Consistent with this is the observation that impaired recruitment of bone marrow-derived stem cells interferes with successful angiogenesis.48 Nevertheless, in the present model, transplanted adult stem cells play a pivotal role in improved and possibly accelerated tissue regeneration with less mortality. This is of particular note because the colonic mucosa already has a very high regenerative capacity. It is conceivable that the improved mucosal perfusion supports the function of preexisting intestinal stem cells or at least inhibits stem cell damage in the epithelium.49

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PII: S0016-5085(06)02679-5

doi:10.1053/j.gastro.2006.12.029

Refers to article:

  • Bone Marrow Stem Cell–Mediated Regeneration in IBD: Where Do We Go From Here?

    Mairi Brittan, Malcolm R. Alison, Stephanie Schier, Nicholas A. Wright
    Gastroenterology March 2007 (Vol. 132, Issue 3, Pages 1171-1173)

Gastroenterology
Volume 132, Issue 3 , Pages 944-954, March 2007

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