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Normal and Gene-Corrected Dystrophic Epidermolysis Bullosa Fibroblasts Alone Can Produce Type VII Collagen at the Basement Membrane Zone  David T. Woodley,

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Presentation on theme: "Normal and Gene-Corrected Dystrophic Epidermolysis Bullosa Fibroblasts Alone Can Produce Type VII Collagen at the Basement Membrane Zone  David T. Woodley,"— Presentation transcript:

1 Normal and Gene-Corrected Dystrophic Epidermolysis Bullosa Fibroblasts Alone Can Produce Type VII Collagen at the Basement Membrane Zone  David T. Woodley, Tom Atha, Yi Huang, Mei Chen  Journal of Investigative Dermatology  Volume 121, Issue 5, Pages (November 2003) DOI: /j x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Expression of type VII collagen in cultured cells used for skin model. (A) Conditioned media from RDEB keratinocytes (lane 1), RDEB fibroblasts (lane 2), normal human fibroblasts (lane 3), and normal human keratinocytes (lane 4) were concentrated and subjected to 6% SDS–PAGE followed by immunoblot analysis using a polyclonal antibody to either NC1 or MMP-2. MMP2 was included as an internal control of protein quality and loading. The position of full-length 290-kDa type VII collagen is indicated. (B) Immunohistochemistry with an affinity-purified polyclonal antibody to the NC1 domain of type VII collagen. RDEB/KC, parental RDEB keratinocytes; RDEB/FB, parental RDEB fibroblasts; HKC, normal human keratinocytes; NFB, normal human fibroblasts. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Immunofluorescence staining of cocultures with antibody to type VII collagen. Cocultures 4 wk after being raised to the air–fluid interface were snap-frozen in liquid nitrogen and embedded in OCT, and 10-μm sections were cut. The sections were subjected to immunofluorescent labeling using an affinity-purified polyclonal antibody to the NC1 domain of type VII collagen (A,C,D,F) and normal rabbit serum (B,E). (A,B) Cocultures composed of normal human fibroblasts combined with parent RDEB keratinocytes. (D,E) Cocultures composed of normal human fibroblasts combined with normal human keratinocytes. (C) Cocultures composed of parental RDEB keratinocytes and RDEB fibroblasts. F, normal human skin. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Expression of type VII collagen in the cultured cells used for CeSSE skin model. (A) Immunoblot analysis of proteins from conditioned media of parental RDEB fibroblasts (lane 1), normal human fibroblasts (lane 2), and gene-corrected RDEB fibroblasts (lane 3). MMP2 was included as an internal control of protein quality and loading. The position of the full-length 290-kDa type VII collagen is indicated. (B) Immunohistochemistry with an affinity-purified polyclonal antibody to the NC1 domain of type VII collagen. Note the very high efficiency of type VII collagen gene transfer to RDEB cells, which, in their native state, cannot express type VII collagen. RDEB/FB, parental RDEB fibroblasts; RDEB/FB/C7, RDEB fibroblasts transduced with lentiviral expression vector for type VII collagen. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Restoration of type VII collagen to reconstituted DEB skin in vivo. Immunofluorescence staining of reconstituted DEB and control skin grafts was performed with antibodies specific for human type VII collagen (A–D) and laminin 5 (E–H). (A,E) Tissue reconstituted from parental RDEB keratinocytes and RDEB fibroblasts. (B,F) Tissue reconstituted from parental RDEB keratinocytes and gene-corrected RDEB fibroblasts. (C,G) Tissue reconstituted from parental RDEB keratinocytes and normal human fibroblasts. (D,H) Normal control tissue reconstituted from normal human epidermal keratinocytes and normal human dermal fibroblasts. Bars, 200 μm. e, epidermis; d, dermis. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Restoration of anchoring fibrils to reconstituted DEB skin in vivo. Transmission electron microscopy was performed on skin regenerated from gene-corrected RDEB fibroblasts with parent RDEB keratinocytes. Restored anchoring fibrils (AF) measuring 200 nm or more are labeled with arrows in the BMZ. The BMZ also has a well formed lamina lucida (LL), lamina densa (LD), and hemidesmosome (HD). Bars, 200 nm. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Sustained deposition of human type VII collagen at the mouse DEJ after a single intradermal injection of fibroblasts. (A) Immunofluorescence staining of mouse skin intradermally injected with fibroblasts 2 wk after injection was performed with either a monoclonal antibody specific for human type VII collagen (green, panels A–C) or a rabbit polyclonal antibody recognizing both mouse and human type VII collagen (red, panels D-F). (A,D,G) Skin injected with gene corrected RDEB fibroblasts. (B,E,H) Skin injected with normal human fibroblasts. (C,F,I) Skin injected with parental RDEB fibroblasts. Merged images (panels G-I) demonstrate the colocalization of human type VII collagen with mouse type VII collagen in the mouse's BMZ. (B) Skin from mice injected intradermally with gene-corrected RDEB fibroblasts was stained with a monoclonal antibody specific for human type VII collagen. (A,D) Biopsies taken 2 mo after injection; (B,E) biopsies taken 3 mo after injection; (C,F) biopsies taken 4 mo after injection. e, epidermis; d, dermis. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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