Direct induction of retinal ganglion cell-like neurons
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE140128 | GEO | 2020/09/30
REPOSITORIES: GEO
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Project description:Retinal ganglion cells (RGCs) are the projection neurons in the retina that connect the visual sensing tissue to the brain. We found that Ascl1/Brn3b/Isl1 transcription factor combination can quickly and efficiently reprogramming mouse embryonic fibroblasts (MEFs) into retinal ganglion cell-like neurons (iRGCs). Using RNA-seq, we analyzed the transcriptomes of MEFs infected with Ascl1/Brn3b/Isl1-overexpressing viruses on day 2 or day 7 of reprogramming, or the final iRGCs on day 13 of reprogramming.
2020-09-30 | GSE140689 | GEO
Project description:Direct induction of retinal ganglion cell-like neurons
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Project description:Direct induction of retinal ganglion cell-like neurons
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Project description:We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and incubated with THY1 antibody (Miltenyi) before undergoing FACS. THY1 positive and THY1 negative cells were subsequently prepared for single cell RNA sequencing. Single cell suspensions were loaded onto 10X Genomics Single Cell 3' Chips along with the reverse transcription master mix as per the manufacturer's protocol for the Chromium Single Cell 3' v2 Library (10X Genomics; PN-120233), to generate single cell gel beads in emulsion. Libraries were then sequenced on an Illumina HiSeq 2500.
2017-10-07 | E-MTAB-6108 | biostudies-arrayexpress
Project description:Visual information is conveyed from the eye to the brain by distinct types of Retinal Ganglion Cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in the combinatorial code for RGC type specification but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a and/or Brn3b positive RGCs, (ii) Brn3a and/or Brn3b dependent RGC transcripts and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation and synaptogenesis. We reveal a combinatorial code of transcription factors, adhesion molecules and determinants of neuronal morphology that are differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.
2017-04-24 | GSE87647 | GEO
Project description:Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (iPSCs) by the overexpression of ATOH7, BRN3B and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers, such as BRN3A, EBF1, ISL1 and RBPMS. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.
2023-08-04 | GSE236027 | GEO
Project description:Since retinal ganglion cells (RGCs) do not regenerate after injury, RGC replacement therapy could provide an approach to vision restoration in glaucoma and other optic neuropathies. Here we developed a rapid protocol for direct induction of RGC (iRGC) differentiation from human iPSCs and ESCs in less than two weeks, by overexpression of NGN2 in RGC survival-promoting medium supplemented with a Notch inhibitor. Neuronal morphology and neurite growth were observed within one week of induction. Immunostaining and qRT-PCR for characteristic RGC-specific genes confirmed identity. Calcium imaging was used to evaluate iRGCs’ electrophysiologic maturation, and demonstrated GABA-induced excitatory calcium influx characteristic of immature RGCs. Using single cell-RNA sequencing (scRNA-seq) we further delineated iRGCs’ differentiation and compared iRGC transcriptomic profiles to fetal human and retinal organoid-derived RGCs. Unbiased clustering showed high similarities of transcriptomic profiles among iRGCs, early-stage fetal human RGCs and early-stage retinal organoid-derived RGCs. However, for some markers, including BRN3a, BRN3b and NEFL, iRGCs demonstrated expression patterns more similar to fetal RGCs than to retinal organoid RGCs. After intravitreal injection into rodent eyes, transplanted iRGCs survived and migrated into host retinas where they were detected one week and one month after transplant. Prior optic nerve trauma to the recipient host did not significantly enhance or detract from iRGC integration, but iRGCs protected host RGCs from neurodegeneration. Taken together, these data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplant, including after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.
2022-04-10 | GSE200452 | GEO
Project description:RNA-seq analysis from young and pre-glaucomatous DBA/2J retinal ganglion cells and control (age and sex-matched, D2-Gpnmb+) retinal ganglion cells
2017-03-07 | GSE90654 | GEO
Project description:Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genome-wide microarray profiling. Localized mRNA from the isolated growth cones of Xenopus laevis and Mus musculus retinal ganglion cells were subjected to microarray analysis
2010-11-23 | E-GEOD-25166 | biostudies-arrayexpress
Project description:During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types. As these different cell types are born, they progress from newly specified cells to fully differentiated neurons and glia. In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina. Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. These experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types. In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type. Experiment Overall Design: Single retinal cells were isolated in tubes containing lysis buffer, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software. Cells were identified post hoc as either developing retinal ganglion cells, amacrine cells or rod photoreceptor cells.
2008-06-16 | E-GEOD-9812 | biostudies-arrayexpress