1. Volume 15, Issue 3, Pages 628-635 (March 2007)
Intravenously Injected Human Fibroblasts Home to Skin Wounds, Deliver Type VII Collagen, and Promote Wound Healing 
David T Woodley, Jennifer Remington, Yi Huang, Yingping Hou, Wei Li, Douglas R Keene, Mei Chen 
Molecular Therapy 
Volume 15, Issue 3, Pages (March 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Expression of C7 in the cultured human RDEB fibroblasts used for IV injection. (a) Conditioned media from RDEB fibroblasts (lane 1), gene-corrected RDEB fibroblasts (lane 2), and normal human fibroblasts (lane 3) were concentrated and subjected to 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot analysis using a polyclonal antibody to NC1. The positions of full-length 290 kDa C7 and molecular weight markers are indicated. (b) Immunohistochemistry with an affinity-purified polyclonal antibody to the NC1 domain of C7. Note the very high efficiency of C7 gene transfer to RDEB cells which, in their native state, cannot express C7. RDEB, parental RDEB fibroblasts; RDEB/C7, RDEB fibroblasts transduced with lentiviral expression vector for C7; NFB, normal human fibroblasts.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
IV injected gene-corrected RDEB fibroblasts delivered human C7 to the mouse's regenerated BMZ. A: (a–h) Immunofluorescence staining of the mouse's skin after IV injection of various types of human fibroblasts was performed with antibodies specific for human C7 at 2 weeks after the injection. Note that the gene-corrected RDEB fibroblasts (a) and normal human fibroblasts (b) synthesized and delivered human C7 to the mouse's regenerated BMZ in wounded sites, but not in unwounded sites (c). Further, there was no human C7 expression in mice (n=10 mice) that were not wounded before injection with either gene-corrected RDEB fibroblasts (d) or normal human fibroblasts (e). Healed wounds of mice injected with parental RDEB cells entirely lacked human C7 (f). (g and h) show the stable expression of human C7 at the mouse's BMZ at 8 weeks after the injection with gene-corrected RDEB fibroblasts and normal human fibroblasts respectively. e, epidermis; d, dermis. B: (i–k) Dose-dependent deposition of human C7 at the mouse's BMZ after IV injection with gene-corrected RDEB fibroblasts. Immunofluorescence staining with an antibody specific for human C7 was performed on healed mouse skin wounds after the animals were injected with (i) 0.75, (j) 1.5, and (k) 3 × 106 gene-corrected RDEB fibroblasts, respectively. Note the dose-dependent increase in the deposition of human C7 at the mouse BMZ. C: Immunofluorescence staining of mouse skin was performed 3 weeks after the animals were IV injected with gene-corrected RDEB fibroblasts (RDEB/C7) or normal human fibroblasts (NFB). The skin was labeled with either a monoclonal antibody specific for human C7 (green, panel α-H) or a rabbit polyclonal antibody that recognizes both mouse and human C7 (red, panel α-H+M). Merged images demonstrate co-localization of human C7 with mouse C7 in the mouse's BMZ. RDEB/C7, gene-corrected RDEB fibroblasts (n=40 mice); NFB, normal fibroblast (n=20 mice); RDEB/FB, parent RDEB fibroblasts (n=20 mice).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Tissue distribution of IV injected gene-corrected RDEB fibroblasts. (a) IV injected gene-corrected RDEB fibroblasts were detected in wounded dermal tissue. Three and 6 weeks after IV injection of gene-corrected RDEB fibroblasts, mouse skin was subjected to double-immunofluorescence labeling with a monoclonal antibody specific for human C7 (α-HC7) and a fluorescein isothiocyanate -conjugated secondary antibody (green) and an antibody specific for human fibroblasts (α-HFB) and Cy3-conjugated secondary antibodies (red). Shown are images from the same field at different wavelengths of excitation and merged. Representative fields of random sections from one of 40 IV injected mice are shown. Merged images (yellow) showed that injected cells were detected within the dermis of the wounded sites. (b) Tissue distribution of IV injected gene-corrected RDEB fibroblasts. Four weeks after IV injection of gene-corrected RDEB fibroblasts, necropsies were performed on the mice (n=20 mice) and tissue sections obtained from brain, kidney, liver, lung, spleen, heart, small intestine, and healed skin were subjected to immunostaining using an antibody specific for human fibroblasts. Note that the injected fibroblasts were readily detected in the wounded skin and in small foci of lung, but not in any other organs.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Immunoelectron microscopy of mouse skin injected with gene-corrected RDEB fibroblasts. Immunogold labeling of mouse skin after IV injection with gene-corrected RDEB fibroblasts was performed using a human-specific anti-C7 antibody and revealed that human C7 delivered via the injected cells incorporated into the mouse's BMZ and formed anchoring fibrils. Note numerous gold particles decorating the ends of anchoring fibrils. Arrows denote gold particle-labeled NC1 domains of human C7 derived from injected fibroblasts. D, dermis; E, epidermis; HD, hemidesmosome. Bar=200 nm.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
IV injected gene-corrected RDEB fibroblasts promoted wound healing in mouse skin. A 1.0-cm2 (1 × 1 cm) full-thickness excision wound was made on the mid-back of 8- to 10-week-old athymic nude mice, which were then injected 8 h after wounding with gene-corrected RDEB fibroblasts expressing human C7 or uncorrected RDEB fibroblasts (n=10 mice per group). (a) Representative days 0, 7, 9, 11, 14, and 17 wounds are shown. Wound sizes were significantly reduced in mice injected with gene-corrected RDEB fibroblasts expressing C7 (RDEB/C7) compared with the mice that were injected with uncorrected RDEB fibroblasts (Control) at 7, 9, and 11 days after wounding. (b) Mean±SD wound size measurements at day 0, 7, 9, 11, 14, and 17 post-wounding (n=10 mice for each group).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
IV injected gene-corrected RDEB fibroblasts promoted wound healing in human skin grafted onto mice. A 0.5-cm2 (8 mm diameter punch biopsy) full-thickness excision wound was made in human skin grafted onto the mid-back of 8- to 10-week-old athymic nude mice, which were then injected 8 h after wounding with gene-corrected RDEB fibroblasts expressing human C7 or without injection (n=5 mice per group). (a) Representative days 0, 5, 8, and 11 wounds are shown. Wound sizes were significantly reduced in mice injected with gene-corrected RDEB fibroblasts expressing C7 (RDEB/C7) compared with control at 5, 8, and 11 days after wounding. (b) Mean±SD wound size measurements at days 0, 5, 8, 11, 15, and 20 post-wounding (n=5 mice for each group).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions