Marrow-Derived Stromal Cell Delivery on Fibrin Microbeads Can Correct Radiation- Induced Wound-Healing Deficits  Michael W. Xie, Raphael Gorodetsky, Ewa D. Micevicz, Natalia C. Mackenzie, Elena Gaberman, Lilia Levdansky, William H. McBride  Journal of Investigative Dermatology  Volume 133, Issue 2, Pages 553-561 (February 2013) DOI: 10.1038/jid.2012.326 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effects of whole-body irradiation (WBI) and skin-only irradiation (SI) on wound healing in C3H mice. (a) Wound tensile strength (WTS) was measured 2 weeks after varying doses of WBI exposure. (b) WTS was measured at 2 weeks after varying doses of SI exposure. (c) The effect of time on gain in WTS was assessed in unirradiated skin (triangles) and skin exposed to 4Gy WBI+21Gy SI (squares). WTS measurements at 2 weeks are unreliable within these high SI doses, as the first “phase” of wound healing is most affected by radiation. (d) WTS was measured 4 weeks after 6Gy WBI or 15Gy SI, or both combined. Error bars indicate mean±SEM, n=4. Significance: *P<0.05, **P<0.01 by Student’s t-test. Journal of Investigative Dermatology 2013 133, 553-561DOI: (10.1038/jid.2012.326) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Phenotypic profile of C57Bl/6 marrow stromal cell-fibrin microbead (FMB). (a, b) Flow-cytometric analysis of mesenchymal marrow-derived stromal cell (MSC) markers Sca1, CD49e, CD44, and CD105 of MSCs grown on FMBs or on plastic. (c, d) MSCs/green fluorescent proteins (GFPs) grown on plastic or loaded on FMBs were implanted in preirradiated wounds in C57Bl/6 mice. Bar=200μm (e) Four weeks after implantation, MSCs/GFP-FMBs were still clearly detectable in healing wounds but not in skin wounds implanted with MSCs isolated and grown on plastic (not shown). Combination of fluorescence with dim light to demonstrate the fluorescence with the outline structures of the intact skin. FSC-H, forward scatter; SI, skin-only irradiation; SSC-H, side scatter; WBI, whole-body irradiation. Bar=200mm. Journal of Investigative Dermatology 2013 133, 553-561DOI: (10.1038/jid.2012.326) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Mesenchymal marrow-derived stromal cell (MSC)-fibrin microbeads (FMBs) correct deficits in gain in wound tensile strength (WTS) caused by whole-body irradiation (WBI). (a) Syngeneic MSC-FMBs were implanted in preirradiated C57Bl/6 mice, and deficit reduction in WTS was assessed after 4 weeks of healing. Mice were exposed to either 18Gy skin-only irradiation (SI) or to 6Gy WBI. FMBs alone were implanted as a control. (b) 1 × 106 MSC-FMBs and 2 × 106 MSC-FMBs were implanted in preirradiated wounds of C57Bl/6 and C3H mice, respectively. Deficit reduction in WTS was assessed after 4 weeks of exposure to 6Gy WBI+15Gy SI. Error bars indicate mean±SEM, n=4. Significance: *P<0.05, **P<0.01 by Student’s t-test. TD, total deficit. Journal of Investigative Dermatology 2013 133, 553-561DOI: (10.1038/jid.2012.326) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Mesenchymal marrow-derived stromal cell (MSC)-fibrin microbead (FMB) implantation times and hair regrowth. (a) 1.5 × 106 MSC-FMBs were implanted into C3H mice wounds immediately after 2 days of loading onto FMBs or after being expanded for a further 5 days in vitro. C3H mice were preirradiated with 4Gy whole-body irradiation (WBI)+21Gy skin-only irradiation (SI) and wound tensile strength (WTS) was assessed after 4 weeks of healing. Error bars indicate mean±SEM, n=4. Significance: *P<0.05, **P<0.01 by Student’s t-test. (b) Skin in C3H mice was irradiated with 4Gy WBI+21Gy SI and was implanted with MSC-FMB regrew hair by 3 weeks after treatment, although color was not restored. TD, total deficit. Journal of Investigative Dermatology 2013 133, 553-561DOI: (10.1038/jid.2012.326) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Hematopoietic mobilization, collagen sheets, and allogeneic and syngeneic mesenchymal marrow-derived stromal cells (MSCs). (a) Five daily injections of granulocyte colony-stimulating factor (G-CSF) were given subcutaneously to previously wounded C3H mice preirradiated with 4Gy whole-body irradiation (WBI)+21Gy skin-only irradiation (SI). An additional single injection of plerixafor was administrated on day 6. (b) MSCs on haptized collagen sheets were implanted in the wound of C3Hf mice previously exposed to 4Gy WBI+21Gy SI. Deficit reduction in wound tensile strength (WTS) was measured 4 weeks after treatment. (c) C3H and C57Bl/6 mice were exposed to 4Gy WBI+21Gy SI, allogeneic or syngeneic MSC+FMBs were implanted in skin wounds, and WTS was assessed at 5 weeks. Error bars indicate mean±SEM, n=4. Significance: *P<0.05, **P<0.01 by Student’s t-test. MSC-Col-Strip, MSC collagen strip; TD, total deficit. M-F is MSC-FMB. Journal of Investigative Dermatology 2013 133, 553-561DOI: (10.1038/jid.2012.326) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions