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REST suppression mediates neural conversion of adult human fibroblasts via microRNA dependent and independent pathways

ABSTRACT: Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs) was achieved for the first time five years ago. This technology offers a promising shortcut for obtaining patient and disease specific neurons for disease modeling, drug screening and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient derived material for large scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron specific microRNAs miR-9 and miR-124, we show that the effect of RESTi is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that RESTi activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes not activated via microRNA overexpression. Based in this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson’s, Huntington’s as well as Alzheimer’s disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE90068 | GEO | 2017/06/03

SECONDARY ACCESSION(S): PRJNA354375

REPOSITORIES: GEO

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Transcriptome profiling of microRNA-mediated neuronal reprogramming with REST repression at day 7

Project description:Ectopic expression of neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. We found that miR-9/9* and miR-124 led to the repression of REST, a transcriptional repressor of neuronal genes, during microRNA-mediated neuronal conversion and knockdown of REST enhanced the activation of BAF53b, a mature neuronal marker. Furthermore, time series analysis of the transcriptome of cells undergoing the miR-9/9*-124-induced conversion indicated upregulated genetic pathways that were predicted to be targeted by REST although the transcript level of REST remained unchanged (Abernathy et al., 2017). Therefore, we reasoned that knocking down REST in addition to miR-9/9*-124 at an early time point (day 7), in which REST repression is minimally evident during neuronal conversion, would speed up the adoption of neuronal identity. We performed the RNA-seq analysis to compared differentially expressed genes (DEGs) between human adult fibroblasts expressing control shRNA (shCTL) and reprogramming cells expressing shCTL or shREST at day 7. Finally, we found that the repression of REST constitutes an important component of microRNA-mediated neuronal reprogramming of human fibroblasts.

2018-05-17 | GSE114541 | GEO

Dual modulation of neuron specific microRNAs and the REST complex promotes functional maturation of induced human adult neurons

Project description:Direct neural reprogramming can be achieved using different approaches, including by expressing neuronal transcription factors or microRNAs, and by knocking down neuronal repressive elements. However, there still exists a high variability in terms of the quality and maturity of the induced neurons (iNs) obtained, depending on the reprogramming strategy employed. Here, we evaluate different long-term culture conditions and study the effect of expressing the neuronal-specific microRNAs miR124 and miR9/9* while reprogramming with Ascl1, Brn2 and knockdown of the neuronal repressor REST. We show that addition of microRNA supports neuronal maturation both in terms of gene and protein expression, as well as promotes the development of active ion channels and the ability of iNs to generate current induced and spontaneous action potentials.

2019-10-01 | GSE132154 | GEO

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages

Project description:Fibroblasts can be reprogrammed to induced neurons (iNs) via transducion of Ascl1, Brn2, and Myt1l. To understand the roles of the circadian regulators Per1 and Per2 on the reprogramming, embryonic fibroblasts from wild type (WT), Per1-/-, and Per2-/- mice were transduced with the three genes and cells were harvested for scRNA-seq on days 4, 7, and 10 after transduction.

2023-11-09 | GSE227621 | GEO

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages

Project description:Fibroblasts can be reprogrammed to induced neurons (iNs) via transducion of Brn2, Ascl1, and Myt1l (BAM). To understand the roles of the circadain regulators Per1 and Per2 on the reprogramming, embryonic fibroblasts from wild type (WT), Per1-/-, and Per2-/- mice were transduced with the three genes and the cells were harvested on day 0, 2, and 4 after transduction for RNA-seq.

2023-11-09 | GSE227140 | GEO

RNA-seq of AD patient-derived induced neurons treated with shikonin

Project description:We have obtained fibroblast cultures from old adult human control donors and Alzheimer patients. The fibroblasts were reprogrammed into directly induced neurons (iNs) to serve as an adult-like and age-equivalent model for aging and neurodegeneration. iNs were treated for 10 days with 10 µM shikonin or DMSO.

2022-07-02 | E-MTAB-11855 | biostudies-arrayexpress

Metabolic changes in Alzheimer patient-derived induced neurons versus non-demented controls

Project description:We have obtained fibroblast cultures from old adult human non-demented control donors and Alzheimer patients (AD). The fibroblasts were reprogrammed into directly induced neurons (iNs) to serve as an adult-like and age-equivalent model for aging and neurodegeneration. Metabolomic landscape and glucose flux in control versus AD were assessed.

2022-06-22 | ST002213 | MetabolomicsWorkbench

The effects of PKM2 modulation and hypoxia on the metabolic landscape of Alzheimer patient-derived induced neurons

Project description:We have obtained fibroblast cultures from old adult human non-demented control donors and Alzheimer patients (AD). The fibroblasts were reprogrammed into directly induced neurons (iNs) to serve as an adult-like and age-equivalent model for aging and neurodegeneration. Their response to PKM2 modulation (shikonin 10 µM or PKM2 overexpression) and hypoxia (CoDo treatment) were assessed.

2022-06-24 | ST002214 | MetabolomicsWorkbench

Direct reprogramming of spiral ganglion non-neuronal cells into neurons: Towards ameliorating sensorineural hearing loss by gene therapy

Project description:The loss of primary auditory neurons (PANs) leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency and these iNs expressed several key markers of neuron identity. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, demonstrating that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.

2018-02-27 | GSE107461 | GEO

Diverse reprogramming codes for neuronal identity

Project description:The transcriptional programs that establish neuronal identity evolved to produce a rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression of certain transcription factors (TFs) can also endow non-neural cells with neuronal properties. To decipher the relationship between reprogramming factors and transcriptional networks that produce neuronal identity and diversity, we screened ~600 TF pairs and identified 76 that produce induced neurons (iNs) from fibroblasts. By intersecting the transcriptomes of iNs with those of endogenous neurons, we define a “core” cell-autonomous neuronal signature. The iNs also exhibit diversity; each TF pair produces iNs with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct TF input “codes” to defined transcriptional outputs, this study uncovers cell autonomous features of neuronal identity and expands the reprogramming toolbox to enable more facile engineering of induced neurons with desired patterns of gene expression and related functional properties.

2018-05-10 | GSE112381 | GEO

Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated microRNA Circuits

Project description:The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. Examination of PTB regulated AGO2/microRNA targeting in Hela cells by CLIP-seq (two biological replicates) , paired-end RNA-seq (control and PTB knockdown) and 3’end stability RNA-seq (control and PTB knockdown)

2013-01-17 | E-GEOD-42701 | biostudies-arrayexpress

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