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.

Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes.

Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

Altered balance of epidermis-related chemokines in epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a congenital, refractory skin disease and there are no fundamental treatments. Recently, allogenic cell therapies are beginning to be applied as potential treatments, that are based on the concept that the allogenic cells can migrate into the skin and reconstitute the skin components. Although the mechanisms of cell migration into skin are not fully understood, chemokines are regarded as key factors in recruiting bone marrow-derived cells. OBJECTIVES: Our study aims to elucidate the expression of chemokines in the EB patients. METHODS: We determined the expression of wound-healing related chemokines in the sera, keratinocytes, and skin tissues of EB patients and compared them to those of healthy volunteers by enzyme-linked immunosorbent assays, quantitative reverse transcription PCR, and immunofluorescence staining. RESULTS: The serum levels of CXCL12 and HMGB1 were found to be significantly elevated in the EB patients. Conversely, the serum levels of CCL21 were found to be lower in the EB patients than in healthy controls. In addition, the serum levels of CXCL12 tended to increase and the serum levels of CCL27 tended to decrease with an increase in the affected body surface areas. To detect the origin of the circulating chemokines, we performed immunofluorescence staining. CCL21, CCL27, HMGB1 and CXCL12 were stained more broadly in the EB patient tissues than those in the control tissues. CONCLUSIONS: These results suggest that fluctuations in chemokine levels may contribute in a coordinated way to the wound-healing process and lend clues toward efficient cell therapies for EB.

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.