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. 2015 Mar 3;5:8718. doi: 10.1038/srep08718

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Copyright © 2015, Macmillan Publishers Limited. All rights reserved

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(A) The experimental design is depicted for in vivo and in vitro experiments (drawn by Hao Chen using Coreldraw X5 and Photoshop CS3 software). For the in vivo experiments, adult Sprague-Dawley rats were not irradiated (control) or were irradiated (IR) with 14 Gy abdominal irradiation at day O, followed by a course of i.v. injection and continuous peritoneal infusion via Alzet pump with control medium (DMEM-F12) or conditioned medium from fibroblasts activated under radiation-induced inflammatory condition (FB-CM^IEC-6(IR)), non-activated MSCs (MSC-CM^IEC-6(NOR)), MSCs activated under radiation-induced inflammatory condition (MSC-CM^IEC-6(IR)) with or without neutralizing antibodies (NAs), MSCs activated by TNF-α, IL-1β and NO donor (MSC-CM^{TNF-α + IL-1β + NO}) and HO-1-knockdown MSCs activated by TNF-α, IL-1β and NO donor (HO1-KD-MSC-CM^{TNF-α + IL-1β + NO}). Rats were either sacrificed at 1, 3, 7 days for structural and functional examination or were monitored for survival status throughout the 14 day course of the experiment. For in vitro experiments, control rat intestinal epithelial IEC-6 cells or cells irradiated with 10 Gy were cultured for 7 days. In parallel, irradiated IEC-6 cells were co-cultured in six-well plates with unconcentrated MSC-CM^IEC-6(NOR) or MSC-CM^IEC-6(IR) with or without neutralizing antibodies, and the proliferation and apoptosis were evaluated every other day as shown. (B) Morphological features of BM-MSCs. (upper panel) Spindle-shaped morphology of primary BM-MSCs at day 3. (lower panel) More confluent BM-MSCs at passage 2. (C) Flow cytometric characterization of BM-MSCs. BM-MSCs (red) expressed CD29 (99.17 ± 1.36%) and CD44 (98.24 ± 1.01%) but not CD34 (2.79 ± 0.24%) and CD45 (1.69 ± 0.19%). (D) MSC-CM^IEC-6(IR) improves the survival rates and lengthens the survival time of rats following 14 Gy abdominal irradiation. (left panel) Cumulative survival for irradiated rats infused with DMEM-F12, FB-CM^IEC-6(IR), MSC-CM^IEC-6(NOR) or MSC-CM^IEC-6(IR) was analyzed using the Kaplan-Meier method. The cumulated number of rats in each experimental group is presented in parenthesis. P-values were determined by log-rank testing. (right panel) Mean survival time. Data represent the mean ± SD. **, P < 0.05 versus IR + DMEM-F12 and IR + FB-CM^IEC-6(IR). ##, P < 0.05 versus IR + MSC-CM^IEC-6(NOR).