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, Véronique Weiler, Peter J. Nelson, Maurice N. Khalil, Sabine Moosmann, Wolf E. Mutschler, Matthias Siebeck, Ralf Huss  Gastroenterology  Volume 132, Issue 3, Pages 944-954 (March 2007) DOI: 10.1053/j.gastro.2006.12.029 Copyright © 2007 AGA Institute Terms and Conditions

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. Gastroenterology 2007 132, 944-954DOI: (10.1053/j.gastro.2006.12.029) Copyright © 2007 AGA Institute Terms and Conditions

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. Gastroenterology 2007 132, 944-954DOI: (10.1053/j.gastro.2006.12.029) Copyright © 2007 AGA Institute Terms and Conditions

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. Gastroenterology 2007 132, 944-954DOI: (10.1053/j.gastro.2006.12.029) Copyright © 2007 AGA Institute Terms and Conditions

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. Gastroenterology 2007 132, 944-954DOI: (10.1053/j.gastro.2006.12.029) Copyright © 2007 AGA Institute Terms and Conditions

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. Gastroenterology 2007 132, 944-954DOI: (10.1053/j.gastro.2006.12.029) Copyright © 2007 AGA Institute Terms and Conditions