Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa.
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ABSTRACT:
SUBMITTER: Tolar J
PROVIDER: S-EPMC3989384 | biostudies-literature | 2014 May
REPOSITORIES: biostudies-literature
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Project description:Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.
| S-EPMC4151825 | biostudies-literature
Project description: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, patient-specific 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.
| S-EPMC3102348 | biostudies-literature
Project description:Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.
| S-EPMC4428910 | biostudies-literature
Project description:BackgroundRevertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes.ObjectivesTo determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions.MethodsSkin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined.ResultsKeratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells.ConclusionsIn documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.
| S-EPMC6766431 | biostudies-literature
Project description:BackgroundRecessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans.MethodsBetween October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography.ResultsOne patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies.ConclusionsIncreased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)
| S-EPMC2967187 | biostudies-literature
Project description:BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB‑c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.
| S-EPMC8663784 | biostudies-literature
Project description:Seven sets of SILAC-based AP-MS experiments were performed analyzing phosphotyrosine signaling after 0, 5, and 10 min of PDGF-BB treatment of primary human skin fibroblasts. For this, proteins phosphorylated on tyrosine residues were enriched using the 4G10 antibody.
2021-11-01 | PXD022985 | Pride
Project description:Small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain. Whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause neuropathic pain is still unclear. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. Damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. It is increasingly recognized that most RDEB patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or neuropathic pain. In this study we investigated the incidence of neuropathic pain and examined the presence of nerve dysfunction in RDEB patients. Around three quarters of patients presented with pain of neuropathic characteristics, which had a length-dependent distribution. Quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). Nerve conduction studies showed normal large fibre sensory and motor nerve conduction; however, skin biopsy showed a significant decrease in intraepidermal nerve fibre density. Autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. We conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length-dependent distribution resulting in disabling neuropathic pain. These findings also support the use of neuropathic pain screening tools in these patients and treatment algorithms designed to target neuropathic pain.
| S-EPMC5405236 | biostudies-literature
Project description:Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.
| S-EPMC5675435 | biostudies-literature
Project description:BackgroundAutosomal recessive dystrophic epidermolysis bullosa (RDEB) is an incurable and severe inherited skin disorder characterized by recurrent blistering at the sublamina densa beneath the cutaneous basement membrane. It is caused by biallelic loss-of-function mutation in the gene encoding type VII collagen (COL7A1). This study aimed to identify the causative variants of a Chinese RDEB patient and further provide prenatal diagnosis for the ongoing risk pregnancy of the proband's mother.MethodsClinical exome sequencing (CES) has been performed and an in-house pipeline was used to conduct a phenotype-driven data analysis. A minigene assay was used to verify the pathogenicity of a novel splice site variant in the COL7A1.ResultsHere we report two compound heterozygous variants in COL7A1, c.3867delT (p.G1290Efs*35) and c.5532+4_5532+5delAG, identified in a RDEB patient by CES. The minigene assay confirmed that thec.5532+4_5532+5delAGchange was a noncanonic splice site variant leading to in an in-frame deletion of exon 64. Prenatal diagnosis indicated that the present pregnancy of the patient's mother was not affected.ConclusionOur study expands the mutation spectrum of COL7A1 and demonstrated that CES and minigene assays were efficient tools for RDEB molecular diagnoses.
| S-EPMC7434731 | biostudies-literature