Volume 18, Issue 4, Pages (April 2016)

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Volume 18, Issue 4, Pages 536-545 (April 2016) Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion Rudolf K. Braun, Jill M. Koch, Timothy A. Hacker, David Pegelow, Jaehyup Kim, Amish N. Raval, Eric G. Schmuck, Denise J. Schwahn, Derek J. Hei, John M. Centanni, Marlowe Eldridge, Peiman Hematti Cytotherapy Volume 18, Issue 4, Pages 536-545 (April 2016) DOI: 10.1016/j.jcyt.2016.01.010 Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 1 Overall study design and time points. Study schema depicting study procedures and assessment time points. BLM, bleomycin instillation; IV, intravenous infusion; PFT, pulmonary function test. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 2 Change in animal weights over the course of the study. All bleomycin-treated animals initially lost weight as a result of the bleomycin. Weight gain steadily increased in all groups after day 8 and continued until the PFT on day 14. Group 1, Blm/vehicle control; group 2, Blm/low-dose MSC; group 3, Blm/high-dose MSC; group 4, control (representative normal healthy animals with no bleomycin exposure). B, Blm instillation; Blm, bleomycin; C, pre-Blm instillation; IV, intravenous infusion; P, pulmonary function test. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 3 Graphic depiction of FEV0.1 (mL/0.1 second) at days 0, 7 and 14. Group 1, Sal/vehicle (representative normal healthy animals with no bleomycin exposure); group 2, Blm/vehicle; group 3, Blm/low-dose MSC; group 4, Blm/high-dose MSC. Data presented at baseline or day 0 (A), day 7 (B) and day 14 (C). Blm, bleomycin; Sal, sterile isotonic saline. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 4 TLC in milliliters at days 0, 7 and 14. Group 1, Sal/vehicle (representative normal healthy animals with no bleomycin exposure); group 2, Blm/vehicle; group 3, Blm/low-dose MSC; group 4, Blm/high-dose MSC. Data presented at baseline or day 0 (A), day 7 (B) and day 14 (C). Blm, bleomycin; Sal, sterile isotonic saline. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 5 Change in SpO2 values during the first (A) and second (B) intravenous infusion. (A) After 20, 25 and 30 min, there was a significant difference in saturated blood oxygen levels between the Blm/low-dose MSC and the Blm/vehicle control groups (Ps = 0.038, 0.038 and 0.019, respectively). No other significant differences were detected between other time points or groups. (B) After the second intravenous infusion, there were no significant differences in saturated blood oxygen levels either within groups or between groups at any of the time points evaluated. Group 1, Blm/vehicle control; group 2, Blm/low-dose MSC; group 3, Blm/high-dose MSC. Blm, bleomycin; P, pre-bleomycin instillation. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 6 Systolic and diastolic RV pressure during second intravenous infusion. There were no significant differences in systolic (A) or diastolic (B) blood pressure at the time of the second intravenous infusion either within or between groups at any of the time points evaluated. Group 1, Blm/vehicle control; group 2, Blm/low-dose MSC; group 3, Blm/high-dose MSC. Blm, bleomycin; P, pre-bleomycin instillation. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions

Figure 7 Pulmonary histology of injured and healthy lungs. Histological comparison of lung tissue either exposed to bleomycin only (left column), bleomycin plus high cell dose (middle column) or representative healthy animal control (right column). Patchy areas of interstitial inflammation, edema and fibrosis are similar in rat lungs treated with bleomycin (A) and bleomycin plus cells (B). Areas of airway dilation (bronchiectasis, b) are similarly rimmed with inflammation in rat lungs treated with bleomycin (D) and bleomycin plus cells (E). A small area of acute alveolar hemorrhage (h) is present in E. The inflammation consists of lymphocytes, histiocytes and neutrophils (n) in G within an edematous (clear) interstitium in both lungs treated with bleomycin (G) and bleomycin plus cells (H). A small amount of acute hemorrhage (h) is present in E and H. Fibrosis (poorly cellular, eosinophilic extracellular material) (f) is common and similar in inflamed areas of rat lungs treated with bleomycin (J) and bleomycin plus cells (K). Panels C, F, I and L are form a healthy unmanipulated, nonstudy rat and are provided for comparison to normal rat lung. Cytotherapy 2016 18, 536-545DOI: (10.1016/j.jcyt.2016.01.010) Copyright © 2016 International Society for Cellular Therapy Terms and Conditions