Intraperitoneal but Not Intravenous Cryopreserved Mesenchymal Stromal Cells Home to the Inflamed Colon and Ameliorate Experimental Colitis

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. RESULTS: Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. CONCLUSIONS: Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis

Migration of intraperitoneally and intravenously administered MSCs in experimental animals.

<p>Tec-99m-labeled MSCs were administered to animals and followed for 24 hours using a gamma-camera. Tec-99m-labeled MSCs were injected intraperitoneally in the right lower abdominal quadrant, and migrated towards the inflamed colon on the contra-lateral left lower quadrant, but not to the non-inflamed colon of controls. Skin fibroblasts used as additional controls did not migrate towards the inflamed colon (A). Tec-99m-labeled MSCs injected through the jugular vein accumulated first into the lungs and then gradually migrated towards liver, spleen, kidneys, and bladder. After 24 hours, labelled cells were barely detectable (B). Images are representative of 3 independent experiments. All animals are lying on their backs. Right (R); left (L).</p

Effect of MSCs on apoptotic rates within the colon.

<p>Apoptotic cells were detected by the TUNEL assay. Photomicrographs of the colon show representative samples of a control, and from TNBS-induced colitic animals treated with BM- and AT-MSCs, or vehicle, and an internal negative control without TdT enzyme (original magnification ×100) (A). Percentages of apoptotic cells in the colonic epithelium and in the lamina propria were analyzed in at least 10 different areas per tissue section. Epithelial values of control and MSCs-treated animals were lower compared to vehicle-treated colitis (p<0.008). Lamina propria values of MSCs- or vehicle-treated animals were higher compared with the control group (p<0.04). Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, and vertical bars represent ranges of 10 animals/group. Differences were analyzed using ANOVA on ranks with Dunnett's test (B).</p

Biological characterization of mesenchymal stromal cells.

<p>Phenotypic analysis of MSCs was carried out by flow cytometry, which revealed that BM- and AT-MSCs expressed the cell markers CD90 (Thy-1) and CD29, but did not express lineage markers such as CD45, CD11b and CD34 (A–F). Solid black lines show AT-MSC, dotted black lines show BM-MSC, and gray lines are isotype control. Functionally, MSCs have the capacity to form different cell lineages. AT- (G–I) and BM- (J–K) MSCs were able to differentiate into adipocytes (H, K) and osteocytes (I, L). These cells were used in subsequent experiments. Oil Red O (G–H, J–K) and Von Kossa (I, L) staining. Length bars represent 50 µm.</p