Cultured Epidermal Autografts from Clinically Revertant Skin as a Potential Wound Treatment for Recessive Dystrophic Epidermolysis Bullosa.

Inherited skin disorders have been reported recently to have sporadic normal-looking areas, where a portion of the keratinocytes have recovered from causative gene mutations (revertant mosaicism). We observed a case of recessive dystrophic epidermolysis bullosa treated with cultured epidermal autografts (CEAs), whose CEA-grafted site remained epithelized for 16 years. We proved that the CEA product and the grafted area included cells with revertant mosaicism. Based on these findings, we conducted an investigator-initiated clinical trial of CEAs from clinically revertant skin for recessive dystrophic epidermolysis bullosa. The donor sites were analyzed by genetic analysis, immunofluorescence, electron microscopy, and quantification of the reverted mRNA with deep sequencing. The primary endpoint was the ulcer epithelization rate per patient at 4 weeks after the last CEA application. Three patients with recessive dystrophic epidermolysis bullosa with 8 ulcers were enrolled, and the epithelization rate for each patient at the primary endpoint was 87.7%, 100%, and 57.0%, respectively. The clinical effects were found to persist for at least 76 weeks after CEA transplantation. One of the three patients had apparent revertant mosaicism in the donor skin and in the post-transplanted area. CEAs from clinically normal skin are a potentially well-tolerated treatment for recessive dystrophic epidermolysis bullosa.

The development of induced pluripotent stem cell-derived mesenchymal stem/stromal cells from normal human and RDEB epidermal keratinocytes.

BACKGROUND: Epidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. OBJECTIVES: We aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). METHODS: Keratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3'-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. RESULTS: After their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. CONCLUSIONS: We successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

Establishment of integration-free induced pluripotent stem cells from human recessive dystrophic epidermolysis bullosa keratinocytes.

BACKGROUND: Induced pluripotent stem cell (iPSC) technology enables patient-specific pluripotent stem cells to be derived from adult somatic cells without the use of an embryonic cell source. To date, recessive dystrophic epidermolysis bullosa (RDEB)-specific iPSCs have been generated from patients using integrating retroviral vectors. However, vector integration into the host genome can endanger the biosafety and differentiation propensities of iPSCs. Although various integration-free reprogramming systems have been reported, their utility in reprogramming somatic cells from patients remains largely undetermined. OBJECTIVE: Our study aims to establish safe iPSCs from keratinocytes of RDEB patients using non-integration vector. METHOD: We optimized and infected non-integrating Sendai viral vectors to reprogram keratinocytes from healthy volunteers and RDEB patients. RESULTS: Sendai vector infection led to the reproducible generation of genomic modification-free iPSCs from these keratinocytes, which was proved by immunohistochemistry, reverse transcription polymerase chain reaction, methylation assay, teratoma assay and embryoid body formation assay. Furthermore, we confirmed that these iPSCs have the potential to differentiate into dermal fibroblasts and epidermal keratinocytes. CONCLUSION: This is the first report to prove that the Sendai vector system facilitates the reliable reprogramming of patient keratinocytes into transgene-free iPSCs, providing another pluripotent platform for personalized diagnostic and therapeutic approaches to RDEB.

Dynamic MR findings of ductal carcinoma in situ within a fibroadenoma.

We report magnetic resonance (MR) imaging findings of ductal carcinoma in situ (DCIS) within a fibroadenoma in a 42-year-old woman. Dynamic MR imaging revealed the mass to have 2 components with different kinetics. A nodular area within the mass showed faster initial enhancement followed by earlier washout and was histologically proven to be DCIS. Dynamic MR imaging reflected differences in vascularity between the fibroadenoma and DCIS, and parameter color maps generated from the dynamic data clearly demonstrated the extent of the DCIS.