Patient-Specific Naturally Gene-Reverted Induced Pluripotent Stem Cells in Recessive Dystrophic Epidermolysis Bullosa Jakub Tolar, John A. McGrath, Lily.

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Patient-Specific Naturally Gene-Reverted Induced Pluripotent Stem Cells in Recessive Dystrophic Epidermolysis Bullosa Jakub Tolar, John A. McGrath, Lily Xia, Megan J. Riddle, Chris J. Lees, Cindy Eide, Douglas R. Keene, Lu Liu, Mark J. Osborn, Troy C. Lund, Bruce R. Blazar, John E. Wagner Journal of Investigative Dermatology Volume 134, Issue 5, Pages 1246-1254 (May 2014) DOI: 10.1038/jid.2013.523 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Mosaicism in recessive dystrophic epidermolysis bullosa (RDEB). (a) Blistered skin in a boy with generalized severe RDEB. Arrow points to the area seen in b. (b) Small patch of unaffected skin in the pubic area (arrows). (c) Blistered skin with no detectable expression of type VII collagen. (d) Nonblistered, pubic-area skin from the same individual with strong continuous linear labeling of type VII collagen (red) at the dermal-epidermal junction. Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Bar=50μm. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Revertant induced pluripotent stem cells (iPSCs) from recessive dystrophic epidermolysis bullosa (RDEB) skin. (a) Persistent mRNA expression of OCT4, SOX2, NANOG, LIN28, and DNMT3b, and transient mRNA expression of c-MYC and KLF4, both consistent with fully reprogrammed mutant and revertant iPSCs. (b) Protein expression in mutant iPSCs of embryonic stem cell surface markers TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase (AF), and transcription factors Nanog and OCT3/4. (c) Same embryonic stem cell protein expression panel in revertant iPSCs. Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Lower panels show the corresponding isotype controls. Bar=50μm. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Epigenetic signature of recessive dystrophic epidermolysis bullosa (RDEB) induced pluripotent stem cells (iPSCs). (a) A partially methylated pattern indicative of gene silencing of OCT4 and NANOG promoter sequences in mutant primary cells. (b) An unmethylated pattern in mutant iPSCs consistent with complete reprogramming into iPSCs. (c) A partially methylated pattern in revertant primary cells. (d) An unmethylated pattern in revertant iPSCs. Open circle indicates unmethylated site in the specified amplicon. Filled circle indicates methylated site in the same amplicon. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In vivo pluripotentiality of recessive dystrophic epidermolysis bullosa (RDEB) induced pluripotent stem cells (iPSCs). (a) Mutant iPSCs injected intramuscularly into immune-deficient mice yielded well-differentiated cystic teratomas with a wide array of cell lineages encompassing cells of endodermal, mesodermal, and ectodermal origins. (b) Same in vivo pluripotentiality was observed in teratomas generated from the revertant iPSCs. Hematoxylin–eosin staining. Bar=50μm. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Hematopoietic differentiation of recessive dystrophic epidermolysis bullosa (RDEB) induced pluripotent stem cells (iPSCs). (a) Hematopoietic CD34-expressing stem cells derived from mutant iPSCs. (b) Hematopoietic CD34-expressing stem cells derived from revertant iPSCs. (c) Hematopoietic progenitors from wild-type, mutant, and revertant iPSCs quantified by the colony-formation assay (mean±SEM; wild type, 52±22 colonies; mutant RDEB, 174±15 colonies; and revertant RDEB, 100±17 colonies). The differences between wild-type and mutant cells, and revertant and mutant cells, are statistically significant (P<0.05), whereas the difference between wild-type and revertant cells is not. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Epidermal differentiation of recessive dystrophic epidermolysis bullosa (RDEB) induced pluripotent stem cells (iPSCs). (a) Keratinocytes derived from revertant iPSCs express type VII collagen (red). Original magnification × 40. (b) Three-dimensional skin from teratoma (Figure 4b) with epidermis-like layers of keratin 5–expressing keratinocytes (green) and linear type VII collagen immunoreactivity at the dermal-epidermal junction (red). Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Bar=50μm. Journal of Investigative Dermatology 2014 134, 1246-1254DOI: (10.1038/jid.2013.523) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions