Embryonic stem cells (ESCs) have an unlimited proliferative capacity and extensive differentiation capability. They are an alternative source for regenerative therapies with a potential role in the treatment of several human diseases. The clinical use of ESCs, however, has significant ethical and biological obstacles related to their derivation from embryos and potential for immunological rejection, respectively. These disadvantages can be circumvented by the alternative use of induced pluripotent stem cells (iPSCs), which are generated from an individual's (autologous) somatic cells by exogenous expression of defined transcription factors and have biological characteristics similar to ESCs. In recent years, patientspecific iPSCs have been generated to study disease mechanisms and develop iPSC-based therapies. The development of iPSC-based therapies for skin diseases requires successful differentiation of iPSCs into cellular components of the skin, including epidermal keratinocytes. Here, we succeeded in generating iPSCs not only from normal human fibroblasts but also from fibroblasts isolated from the skin of two patients with recessive dystrophic epidermolysis bullosa. Moreover, we differentiated both of these iPSCs into keratinocytes with high efficiency, and generated 3D skin equivalents using iPSC-derived keratinocytes, suggesting that they were fully functional. Our studies indicate that autologous iPSCs have the potential to provide a source of cells for regenerative therapies for specific skin diseases.reprogramming | retinoic acid | bone morphogenetic protein 4 | cellular therapy
This novel MSA is associated with cancer-associated DM and may serve as a diagnostic serological marker for this specific subset.
Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells.
Revertant mosaicism is a naturally occurring phenomenon involving spontaneous correction of a pathogenic gene mutation in a somatic cell. It has been observed in several genetic diseases, including epidermolysis bullosa (EB), a group of inherited skin disorders characterized by blistering and scarring. Induced pluripotent stem cells (iPSCs), generated from fibroblasts or keratinocytes, have been proposed as a treatment for EB. However, this requires genome editing to correct the mutations, and, in gene therapy, efficiency of targeted gene correction and deleterious genomic modifications are still limitations of translation. We demonstrate the generation of iPSCs from revertant keratinocytes of a junctional EB patient with compound heterozygous COL17A1 mutations. These revertant iPSCs were then differentiated into naturally genetically corrected keratinocytes that expressed type XVII collagen (Col17). Gene expression profiling showed a strong correlation between gene expression in revertant iPSC-derived keratinocytes and the original revertant keratinocytes, indicating the successful differentiation of iPSCs into the keratinocyte lineage. Revertant-iPSC keratinocytes were then used to create in vitro three-dimensional skin equivalents and reconstitute human skin in vivo in mice, both of which expressed Col17 in the basal layer. Therefore, revertant keratinocytes may be a viable source of spontaneously gene-corrected cells for developing iPSC-based therapeutic approaches in EB.
The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.
We report five women who presented with scleroderma due to taxanes, mimicking systemic sclerosis. All five patients had received taxane chemotherapy for the treatment of metastatic breast cancer. Marked oedema began first, followed by skin sclerosis occurring mainly at the distal ends of the extremities 6-12 months after the administration of taxane in all patients. Skin biopsies showed full-layer dermal fibrosis with thickened collagen bundles, and perivascular monocytic cell infiltration. These cases resemble systemic sclerosis in terms of their clinical course and histological findings. However, clinical findings including Raynaud's phenomenon and pulmonary fibrosis as well as immunological abnormalities associated with systemic sclerosis were not detected in any of the patients. Although the mechanisms have not been clarified, it should be noted that taxane is causally involved in the formation of scleroderma-like skin conditions.
Hereditary leukonychia (porcelain nails or white nails) is a rare nail disorder with an unknown genetic basis. To identify variants in a gene underlying this phenotype, we identified four families of Pakistani origin showing features of hereditary leukonychia. All 20 nails of each affected individual were chalky and white in appearance, consistent with total leukonychia, with no other cutaneous, appendageal, or systemic findings. By using Affymetrix 10K chip, we established linkage to chromosome 3p21.3-p22 with a LOD score (Z) of 5.1. We identified pathogenic mutations in PLCD1 in all four families, which encodes phosphoinositide-specific phospholipase C delta 1 subunit, a key enzyme in phosphoinositide metabolism. We then identified localization of PLCD1 in the nail matrix. It was recently shown that PLCD1 is a component of the human nail plate by proteomic analysis and is localized in the matrix of human nails. Furthermore, mutations detected in PLCD1 resulted in reduced enzymatic activity in vitro. Our data show that mutations in PLCD1 underlie hereditary leukonychia, revealing a gene involved in molecular control of nail growth.
Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31 + cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB.
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