Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Sclerodermatous Chronic Graft-versus-Host Disease: Clinical Report Hong Zhou,

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Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Sclerodermatous Chronic Graft-versus-Host Disease: Clinical Report Hong Zhou, Mei Guo, Chunjing Bian, Zhao Sun, Zhuo Yang, Yang Zeng, HuiSheng Ai, Robert Chunhua Zhao Biology of Blood and Marrow Transplantation Volume 16, Issue 3, Pages 403-412 (March 2010) DOI: 10.1016/j.bbmt.2009.11.006 Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Biopsy specimen of chest skin in patient 2 before MSC infusion. Stratified squamous epithelium showing mild hyperkeratosis with parakeratosis, basal cell liquefaction degeneration, and T lymphocytes infiltrating into the superficial layer of the derma, with visible manipulus of tissue cell–phagocytized hemosiderin. (Hematoxylin and eosin staining; original magnification 100×.) Biology of Blood and Marrow Transplantation 2010 16, 403-412DOI: (10.1016/j.bbmt.2009.11.006) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 In vitro differentiation and typical phenotypes of MSCs. (A) Cell morphology of MSCs at the third passage. (B) Oil Red O staining of MSCs after adipogenic induction. (C) von Kossa staining of MSCs after osteogenic induction. (D) Safranin O staining of MSCs after chondrogenic induction. (E) Phenotypes of MSCs at the third passage. Biology of Blood and Marrow Transplantation 2010 16, 403-412DOI: (10.1016/j.bbmt.2009.11.006) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Effect of MSCs on T lymphocyte proliferation in mitogen proliferative assays. Shown are nonstimulated T cells (T0), PHA-stimulated T cells (Ts), and PHA-stimulated T cells cocultured with MSC at a MSC:T cell ratio of 1:10. Data are given as the mean ± standard deviation of 3 independent experiments. ∗P <.05 versus T0; ∗∗P <.05 versus Ts. Biology of Blood and Marrow Transplantation 2010 16, 403-412DOI: (10.1016/j.bbmt.2009.11.006) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 The change process of skin lesions before and after MSC infusion in patient 4. Shown are changes in the skin lesion of the neck (A), left arm (B), back (C), and right ankle (D) on days 0, +14, +28, +33, +40, +54, +76, +92, and +123 after initial MSC infusion. Over time, the skin became soft, desquamation was reduced, skin color was shallowed, edema disappeared, and the ankle ulcer formed a scab and healed. Biology of Blood and Marrow Transplantation 2010 16, 403-412DOI: (10.1016/j.bbmt.2009.11.006) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Percentage of intracellular cytokines producing T lymphocyte subsets detected by flow cytometry. (A) Patient 1: polygram detected by flow cytometry on days 0, +14, +28, and +49 of MSC infusion. (B) Patient 2: polygram detected by flow cytometry on days 0, +14, +39, and +47 of MSC infusion. (C) Patient 3: polygram detected by flow cytometry on days 0, +8, +15, and +22 of MSC infusion. (D) Patient 4: polygram detected by flow cytometry on days 0, +7, +14, +28, and +35 of MSC infusion. (E) Patient 4: histogram of IL-2–, IFN-γ–, IL-10–, and IL4–producing T lymphocyte subsets on days 0 and +35 of MSC infusion. (F) Comparison of the 4 intracellular cytokines producing cells between before infusion of MSCs and the final infusion of MSCs. The difference is statistically significant (∗P <.05). Biology of Blood and Marrow Transplantation 2010 16, 403-412DOI: (10.1016/j.bbmt.2009.11.006) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions