Direct induction of retinal ganglion cell-like neurons
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE140689 | GEO | 2020/09/30
REPOSITORIES: GEO
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Project description:Mouse embryonic fibroblasts were reprogrammed into retinal ganglion cell-like neurons by overexpressing Ascl1/Brn3b/Isl1 transcription factor combination in 13 days.
2020-09-30 | GSE140128 | 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: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:Regeneration of neurons has enormous implications for human health and is a topic of interest both from the biological and translational perspective. The retina poses an excellent system to study the potential of replacing neurons following injury and the impact different lesions can have on this process. We have established mouse models where proneural transcription factors are expressed in Müller glia to stimulate neurogenesis, which is analogous to how zebrafish regenerate the retina after injury. Our previous studies have shown that Müller glia overexpressing either Ascl1 or Ascl1-Atoh1 will respond to inner retinal damage, caused by a neurotoxic dose of NMDA, by acquiring a progenitor-like state and giving rise to bipolar cells or retinal ganglion-like cells, respectively. It was not known however whether neurogenesis would still occur if the retina suffered outer retinal injury and if the timing of injury relative to the expression of proneural factors was critical for the process. To address these questions, we explored whether timing or mode of injury affect the induced neurogenesis from MG. We show that reprogramming MG with Ascl1 is not impacted by the mode or timing of injury, since all paradigms resulted in similar ratios of new bipolar neurons. By contrast, MG that express Ascl1-Atoh1 respond to outer retinal damage by producing a new type of retinal ganglion-like cell that is not generated after NMDA injury. Therefore, although the fate of reprogrammed neurons is mostly dictated by the proneural transcription factors, there is a context-dependent effect of the injured retinal microenvironment.
2023-12-13 | GSE250019 | GEO
Project description:Regenerative neuroscience aims to stimulate endogenous repair mechanisms in the nervous system to replace neurons that have been lost from degenerative diseases or trauma. In the retina, many non-mammalian vertebrate species spontaneously regenerate retinal neurons from glial cells, through an injury-induced reprogramming of the cells to a retinal progenitor state. Recently, we have reported that engineering mouse Muller glia to express a retinal progenitor gene, the proneural transcription factor, Ascl1, is sufficient to stimulate neurogenesis in the glia to generate functional neurons. However, this process is inefficient and only a third of the Ascl1-expressing glia generate new neurons. Here we test whether proneural transcription factors of the Atoh1/7 class can further promote the neurogenic capacity of MG. We report that the combination of Ascl1:Atoh1 is remarkably efficient at stimulating neurogenesis (>80% expressing cells), even in the absence of retinal injury or TSA. We use electrophysiology and scRNA-seq to demonstrate that Ascl1:Atoh1 generates a diversity of retinal neuron types with the majority expressing characteristics of retinal ganglion cells. Overall, our results provide a proof-of-principle that transcription factors that are normally sequentially active in cell fate specification, can when combined, substantially improve glial reprogramming to neurons and expand the repertoire of cell fates that can be regenerated in the adult mouse retina. The ability to stimulate the neurogenic potential of glia in adult mice with high efficiency is a key step towards translation of this approach to repair the central nervous system.
2021-10-19 | GSE184286 | GEO
Project description:Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.
2021-01-10 | GSE164489 | GEO
Project description:Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in non-mammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. In this report, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in MG, including Ascl1, Pou4f2, Islet1 and Atoh1. Using IHC, scRNA-seq, scATAC-seq, and electrophysiology we find retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental RGC-transcription factors.
2022-09-15 | GSE211077 | GEO
Project description:Axon pathfinding is a key step during the formation of neural circuits but however the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline that neurons make during developmente midline that mammalian retinal ganglion cell (RGC) axons take at the optic chiasm has classically represented a robust model to search for novel mechanisms controlling the selection of axonal trajectories. Here, in order to identify new axon pathfinding regulators, we compared the transcriptome and chromatin accessibility profiles of mammalian retinal ganglion cells RGCs projecting ipsilateral (iRGCs) or contralaterally (cRGCs). Overall, our analyses retrieved dozens of new molecules potentially involved in axon pathfinding and uncovered the regulatory logic behind axon trajectories selection.
2022-08-22 | GSE184275 | GEO
Project description:Retinal ganglion cells (RGCs) are the projection neurons of the retina. Proper development of the axon and dendrite of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of a newly identified RGC-specific gene, Shtn1, in neurite development in RGCs. Using IF, we demonstrated that SHTN1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites, and thus the electrophysiological maturation, of iRGCs. RNA-Seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. These findings improve our understanding of the molecular machinery governing RGC neurite development, and may help to optimize future RGC regeneration methods.
2024-07-10 | GSE254839 | GEO
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