1. Potential of Fibroblast Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa 
Tracy Wong, Luke Gammon, Lu Liu, Jemima E. Mellerio, Patricia J.C. Dopping-Hepenstal, John Pacy, George Elia, Rosemary Jeffery, Irene M. Leigh, Harshad Navsaria, John A. McGrath 
Journal of Investigative Dermatology 
Volume 128, Issue 9, Pages (September 2008)
DOI: /jid
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2. Figure 1
Type VII collagen immunolabeling at the DEJ increases following intradermal injection of allogeneic fibroblasts in most RDEB subjects. The pathogenic COL7A1 mutations are listed in the left column and the asterisks indicate the heterozygous mutations present in the respective parent donor fibroblasts. ND=mutation not determined. The type VII collagen antibody staining illustrated is LH7.2. (a–e) Immunolabeling of the subjects’ baseline skin (that is, before fibroblast injections) demonstrates almost absent type VII collagen staining in subjects 1 and 3 (a, c), markedly reduced staining in subject 2 (b), and slight reduced intensity labeling in subjects 4 and 5 (d, e), compared with bright linear staining at the DEJ seen in control skin (f). Autologous fibroblast injections did not lead to any increase in type VII collagen immunostaining compared with baseline at 2 weeks (g–k) or at 3 months (v–z) in any of the five subjects. In contrast, parent donor allogeneic fibroblast injections led to a variable increase in type VII collagen labeling at the DEJ in all individuals at 2 weeks (l–p), which was still present at 3 months for patients 2, 4 and 5 (bb, dd, ee). Unrelated donor allogeneic fibroblast injections also led to an increase in type VII collagen labeling at 2 weeks in subjects 2, 4 and 5 (r, t, u), which was still detectable at 3 months (gg, ii, jj). For parental and unrelated donor allogeneic fibroblasts, the increased staining was most marked in subject 4 (o, t, dd, ii) and subject 5 (p, u, ee, jj). Bar=50μm. Arrows indicate DEJ.
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Fibroblast injection sites and biopsy time points in recessive dystrophic EB skin. Grids measuring 1cm2 were drawn onto intact, non-blistered skin of each subject. Six injections were given as shown and biopsies were then taken either at 2 weeks or at 3 months. Four of the five baseline biopsies were taken from adjacent skin, as illustrated.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
Transmission electron microscopy shows an increase in the number of rudimentary anchoring fibrils 3 months following intradermal injection of allogeneic fibroblasts. This figure illustrates the morphological appearances of the DEJ in skin biopsies performed in subject 4. (a) The ultrastructural appearances of normal skin show anchoring fibrils that insert into the lamina densa (arrows) and that have a fan-shaped appearance and central cross-banding. (b) At baseline, skin of subject 4 demonstrates a few anchoring fibril-like structures extending from the DEJ (arrows). Many of these fibrils appear short, thin, and lack central cross-banding and fan-shaped appearance. (c) Three months following injection of unrelated allogeneic fibroblasts there is an increased number of anchoring fibrils along the DEJ in the skin of subject 4 (arrows), although most of these appear rudimentary and lack the morphologic characteristics of the normal skin anchoring fibrils shown in (a). Bars=0.2μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Immunohistochemical labeling shows only transient inflammation following intradermal injection of allogeneic fibroblasts. This figure illustrates the immunohistologic findings in subject 5 using CD45 antibody (pan-T cell marker) and CD11c (dendritic cell marker). (a) CD45 staining of baseline skin reveals only a few lymphocytes in the dermis. (b) Two weeks following autologous fibroblast injection there is a slight increase in the number of lymphocytes in the papillary dermis. (c) Two weeks after injection of parental fibroblasts and also (d) unrelated allogeneic fibroblasts the number of dermal lymphocytes is increased. However, the infiltrates were barely detectable at three months (data not shown). (e) CD11c staining of baseline skin. Mild infiltration of dendritic cells in the papillary dermis is noted at two weeks following injection of autologous fibroblasts (f), parental fibroblasts (g), and unrelated allogeneic fibroblasts (h). Bars=50μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Fluorescent Y-probe in situ hybridization shows no detectable allogeneic fibroblasts 2 weeks after intradermal injection. (a) Male positive control skin with positively labeled cells indicated by the single peripheral green spot within the 4′-6′-diamidino-2-phenylindole-stained nucleus. (b) In the skin of subject 5 (female), biopsy taken at 2 weeks following injection of allogeneic fibroblasts (male donor) shows no detectable Y-positive cells. Positions of the epidermis and dermis are shown and the dot-dash line indicates the DEJ. Bars=25μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
The recipient's mutant COL7A1 gene expression is increased 3 months after intradermal injection of allogeneic fibroblasts. (a) Reverse transcriptase-PCR for COL7A1 was performed using primers sited in exons 28 and 35 with template cDNA obtained from skin biopsies taken at baseline and following injection of autologous, parental, and unrelated allogeneic donor fibroblasts at 3 months. Normal skin and negative control were also included. Compared with the reduced intensity PCR products at baseline, the corresponding amplification products 3 months following unrelated allogeneic donor fibroblast injections show increased COL7A1 cDNA. GAPDH control for equivalent template loading is shown below. (b) GeneMapping of the amplified normal skin control cDNA reveals only one product size represented by a single peak. In contrast, the baseline amplified cDNA from subject 4 depicts two peaks, the smaller one representing the allele with the single-base deletion from the splice-site mutation IVS30-1G>A, and the other denoting the allele with the missense mutation p.R2069C. Compared with baseline, 3 months following injection of parent and especially unrelated allogeneic fibroblasts, there is an increase in the total expression of COL7A1 as well as an increase in the 1-bp deletion transcript, indicating that the origin of the COL7A1 is derived from the recipient's own keratinocytes/fibroblasts. MW, molecular weight marker; GAPDH, glyceraldehye-3-phosphate dehydrogenase.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions