Volume 140, Issue 3, Pages 966-975.e4 (March 2011) Bone Marrow Stromal Cell Transplants Prevent Experimental Enterocolitis and Require Host CD11b+ Splenocytes  Biju Parekkadan, Rabi Upadhyay, Joshua Dunham, Yoshiko Iwamoto, Emiko Mizoguchi, Atsushi Mizoguchi, Ralph Weissleder, Martin L. Yarmush  Gastroenterology  Volume 140, Issue 3, Pages 966-975.e4 (March 2011) DOI: 10.1053/j.gastro.2010.10.013 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Prevention of trinitrobenzosulfonic acid−induced colitis by MSC transplantation. (A) Kaplan–Meier analysis of cell-based transplantation strategies with intravenous treatment. (B) Percentage of original body weight loss of mice over time. (C) Semi-quantitative fecal occult blood testing of experimental groups. Fb, fibroblast; U, unit or the equivalent of 1 × 106 cells. For prevention studies, the cohorts were: ethanol sham (n = 4), 1 U MSC (n = 16), 0.25 U MSC (n = 8), saline (n = 15), or Fb (n = 6). *P < .05 and **P < .005. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Histopathological analysis of colitic mice after MSC infusion. Mesenteric lymph node (A) and transverse colon (B) of colitic mice 7 days post-cell therapy in the prevention trial. Lymph node cellularity and colon weight per length ratios are stated below the group names in units of [×106 cells/node] and [mg/cm], respectively. Representative microscopic specimens are shown for (C) saline, (D) fibroblast, and (E) MSC-treated colitic mice compared to (F) ethanol sham controls. Pathology scores are stated above the images. Histopathology analysis was performed on n = 4 of each group. EtOH, ethanol (sham control). Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Increased regulatory T-cell number in mesenteric lymph nodes after MSC therapy. Flow cytometry of mesenteric lymph nodes for CD25 and Foxp3 expression. The graph represents 1 of 2 independent trials of n = 4. APC, allophycocyanin; EtOH, ethanol (sham control); Fb, fibroblast; PE, phycoerythrin; U, unit or the equivalent of 1 × 106 cells; WT, wild-type (healthy control). *P < .05. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Generation of Foxp3+ splenocytes after MSC coculture is dependent on CD11b+ Cells. Splenocytes were cultured for 5 days with or without mesenchymal cells at a 1:10 ratio (mesenchymal cell to splenocyte) and analyzed for Foxp3 expression using flow cytometry. Dot plots gated on CD4 expression of unfractionated splenocytes cultured with (A) IL-2 alone, (B) fibroblasts (Fb) and IL-2, or (C) MSCs and IL-2. The graph represents 1 of 5 independent trials. (D) Dot plot of CD11b+ depleted splenocytes cocultured with MSCs and IL-2. (E) Results of 5 independent trials comparing percentage of CD25+ Foxp3+ cells to coculture conditions. APC, allophycocyanin; PE, phycoerythrin. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Near infrared tracking of MSC transplants. MSCs were labeled with VT680 and infused intravenously at the time of trinitrobenzosulfonic acid administration. Control images, labeled as “dye,” are injections of VT680 dye alone. Representative images from the (A) lungs, (B) mesenteric lymph nodes, (C) large intestine, and (D) spleens of mice injected with the dye alone or labeled MSCs 1 day post-infusion. Results of 2 independent imaging studies of n = 3 per group. (E) Immunofluorescent micrographs of splenic tissue harvested from mice infused with VT680-labeled MSCs (red) and co-stained for CD11b (green), and 4′,6-diamidino-2-phenylindole (blue). Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Depletion of CD11b+ cells by liposomal cytotoxicity or splenectomy eliminates the therapeutic effect of MSCs. (A) Kaplan–Meier analysis of TNBS-induced mice depleted of monocytes and macrophages by liposomal clodronate and intravenously treated with saline or MSCs. (B) Total number of CD25+ Foxp3+ cells in mesenteric lymph nodes after treatment of monocytic-depleted mice. (C) Kaplan–Meier analysis of TNBS-induced mice intravenously treated with saline or MSCs that were splenectomized before disease induction. (D) Total number of CD25+ Foxp3+ cells in mesenteric lymph nodes after treatment of splenectomized mice. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Adoptive transfer of CD11b+ splenocytes after MSC coculture confers increase in regulatory T-cell number and survival benefit in colitic mice. (A) Schematic of coculture composition. CD11b+ cells were isolated by magnetic activated cell sorting and directly cocultured with MSCs or fibroblasts for 5 days in vitro. Subsequently, CD11b+ cells were harvested by magnetic separation and were assessed for their immunomodulatory function in vitro and in vivo. (B) Box plots of CD25 and Foxp3 expression of splenocytes after incubation with cocultured CD11b+ cells (ratio denotes mesenchymal cell:splenocyte) for 5 days in regulatory T-cell stimulating conditions. The graph represents 1 of 2 independent trials of n = 3 wells. (C) Survival of TNBS−induced mice after treatment with cocultured CD11b+ cells at a 1-week study end point. n = 10 per group. (D) Total number of CD25+ Foxp3+ cells in mesenteric lymph nodes after treatment of colitic mice with transferred CD11b+ cells. Fb, fibroblast. U, unit or the equivalent of 1 × 106 cells. *P < .05. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Intraperitoneal delivery and therapeutic trials of MSC grafts in acute colitis. (A) Survival analysis of 0.25 U or 1 U MSCs injected intraperitoneally into colitic mice in a preventative setting. (B) Survival analysis and (C) body weight loss in experimental mice after intravenous delivery of cells at 2 days after disease onset. (D) Survival analysis and (E) body weight loss in experimental mice after intravenous delivery of cells at 1 day after disease onset. EtOH, ethanol (sham control); Fb, fibroblast; U, unit or the equivalent of 1 × 106 cells. **P < .005. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Generation of Foxp3+ splenocytes after MSC coculture is independent of enhanced proliferation of CD4+ CD25+ T cells or conversion of naïve cells to a suppressor phenotype. Dot plots gated on CD4 expression of CD4+ CD25− splenocytes were cultured for 5 days (A) with or (B) without MSCs and analyzed for Foxp3 expression using flow cytometry. The graph represents 1 of 3 independent trials of n = 4. (C) Total Foxp3+ cell number after coculture of magnetic activated cell sorting separated CD25+ cells with no cell, MSCs, or in the presence of recombinant human interleukin-2 for 5 days. After culture, cells were counted and analyzed for CD25+ Foxp3+ cells. The product of cell number and percentage of CD25+ Foxp3+ cell number is plotted as the absolute cell number for each condition. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Viability and intracellular fluorescence of MSCs loaded with VT680. (A) MSCs were incubated with VT680 at 0, 3, 30, and 300 mg/mL for 30 minutes on ice and viability was determined using trypan blue exclusion assay. (B) Representative histograms of VT680 expression by MSCs loaded with 0 (gray), 3 (blue), and 30 (red) mg/mL dye. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Splenic CD11b+ cells. (A) Immunofluorescent micrographs of wild-type splenic tissue stained for CD11b (green) and 4′,6-diamidino-2-phenylindole (blue). (B) Histogram of CD11b distribution in cells that were isolated after MSC in vitro coculture. (C) Dot plot of F4/80 and Gr-1 frequency in CD11b+ gated cells after MSC coculture. Gastroenterology 2011 140, 966-975.e4DOI: (10.1053/j.gastro.2010.10.013) Copyright © 2011 AGA Institute Terms and Conditions