Restoring vision and rebuilding the retina by Müller glial cell reprogramming.

Müller glia are non-neuronal support cells that play a vital role in the homeostasis of the eye. Their radial-oriented processes span the width of the retina and respond to injury through a cellular response that can be detrimental or protective depending on the context. In some species, protective responses include the expression of stem cell-like genes which help to fuel new neuron formation and even restoration of vision. In many lower vertebrates including fish and amphibians, this response is well documented, however, in mammals it is severely limited. The remarkable plasticity of cellular reprogramming in lower vertebrates has inspired studies in mammals for repairing the retina and restoring sight, and recent studies suggest that mammals are also capable of regeneration, albeit to a lesser degree. Endogenous regeneration, whereby new retinal neurons are created from existing support cells, offers an exciting alternative approach to existing tissue transplant, gene therapy, and neural prosthetic approaches being explored in parallel. This review will highlight the role of Müller glia during retinal injury and repair. In the end, prospects for advancing retinal regeneration research will be considered.

Greater Outflow Facility Increase After Targeted Trabecular Bypass in Angiographically Determined Low-low Regions.

PURPOSE: To investigate the impact of trabecular bypass surgery targeted to angiographically determined high- vs. low-aqueous humor outflow areas on outflow facility (C) and intraocular pressure (IOP). DESIGN: Ex vivo comparative study. SUBJECTS: Postmortem ex vivo porcine and human eyes. METHODS: Porcine (n = 14) and human (n = 13) whole globes were acquired. In both species, anterior segments were dissected, mounted onto a perfusion chamber, and perfused using Dulbecco's phosphate buffered solution containing glucose in a constant flow paradigm to achieve a stable baseline. Fluorescein was perfused into the anterior chamber and used to identify baseline segmental high- and low-flow regions of the conventional outflow pathways. The anterior segments were divided into 2 groups, and a 5 mm needle goniotomy was performed in either a high- or low-flow area. Subsequently, C and IOP were quantitatively reassessed and compared between surgery in baseline "high-flow" and "low-flow" region eyes followed by indocyanine green angiography. MAIN OUTCOME MEASURES: Outflow facility. RESULTS: In all eyes, high- and low-flow segments could be identified. Performing a 5-mm goniotomy increased outflow facility to a variable extent depending on baseline flow status. In the porcine high-flow group, C increased from 0.31 ± 0.09 to 0.39 ± 0.09 µL/mmHg/min (P = 0.12). In the porcine low-flow group, C increased from 0.29 ± 0.03 to 0.56 ± 0.10 µL/mmHg/min (P < 0.001). In the human high-flow group, C increased from 0.38 ± 0.20 to 0.41 ± 0.20 µL/mmHg/min (P = 0.02). In the human low-flow group, C increased from 0.25 ± 0.11 to 0.32 ± 0.11 µL/mmHg/min (<0.001). There was statistically significant greater increase in C for eyes where surgery was targeted to baseline low-flow regions in both porcine (0.07 ± 0.09 vs. 0.27 ± 0.13, P = 0.007 µL/mmHg/min, high vs low flow) and human eyes (0.03 ± 0.03 vs. 0.07 ± 0.02, P = 0.03 µL/mmHg/min, high vs. low flow). CONCLUSIONS: Targeting surgery to low-flow areas of the trabecular meshwork yields higher overall facility increase and IOP reduction compared to surgery in high-flow areas. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Bulk RNA sequencing analysis of developing human induced pluripotent cell-derived retinal organoids.

Retinogenesis involves the transformation of the anterior developing brain into organized retinal lamellae coordinated by intricate gene signalling networks. This complex process has been investigated in several model organisms such as birds, fish, mammals and amphibians, yet many facets of retinal development are different in humans and remain unexplored. In this regard, human pluripotent stem cell (hPSC)-derived 3D retinal organoids and Next Generation Sequencing (NGS) have emerged as key technologies that have facilitated the discovery of previously unknown details about cell fate specification and gene regulation in the retina. Here we utilized hPSCs integrated with fluorescent reporter genes (SIX6-p2A-eGFP/CRX-p2A-h2b-mRuby3) to generate retinal organoids and carry out bulk RNA sequencing of samples encompassing the majority of retinogenesis (D0-D280). This data set will serve as a valuable reference for the vision research community to characterize differentially expressed genes in the developing human eye.

A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.

Chromatin Accessibility and Transcriptional Differences in Human Stem Cell-Derived Early-Stage Retinal Organoids.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Temporal and Isoform-Specific Expression of CTBP2 Is Evolutionarily Conserved Between the Developing Chick and Human Retina.

Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.