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Purpose: Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods: Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results: Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions: Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance: This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.
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Transportadoras de Casetes de Unión a ATP , Células Epiteliales , Transportadoras de Casetes de Unión a ATP/genética , Humanos , Fenotipo , Pigmentos Retinianos , Enfermedad de StargardtRESUMEN
The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.
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A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.
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Reprogramación Celular/genética , Cromosomas de los Mamíferos/genética , Metilación de ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Cromosoma X/genética , Animales , Sitios de Unión , Islas de CpG/genética , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Genoma , Impresión Genómica , Células Madre Pluripotentes Inducidas/citología , Masculino , Ratones , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. METHODS: We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. RESULTS: Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. CONCLUSIONS: This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.
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Reprogramación Celular/genética , Virus de la Encefalitis Equina Venezolana/genética , Células Epiteliales/efectos de los fármacos , Fibroblastos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Degeneración Retiniana/terapia , Animales , Diferenciación Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/metabolismo , Células Epiteliales/citología , Células Epiteliales/fisiología , Células Epiteliales/trasplante , Fibroblastos/citología , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Inyecciones Intraoculares , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Cultivo Primario de Células , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/fisiología , Piel/citologíaRESUMEN
Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.