An enhancer located in a Pde6c intron drives transient expression in the cone photoreceptors of developing mouse and human retinas.

How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans.

Biophysical, Molecular and Proteomic Profiling of Human Retinal Organoid-Derived Exosomes.

PURPOSE: There is a growing interest in extracellular vesicles (EVs) for ocular applications as therapeutics, biomarkers, and drug delivery vehicles. EVs secreted from mesenchymal stem cells (MSCs) have shown to provide therapeutic benefits in ocular conditions. However, very little is known about the properties of bioreactor cultured-3D human retinal organoids secreted EVs. This study provides a comprehensive morphological, nanomechanical, molecular, and proteomic characterization of retinal organoid EVs and compares it with human umbilical cord (hUC) MSCs. METHODS: The morphology and nanomechanical properties of retinal organoid EVs were assessed using Nanoparticle tracking analysis (NTA) and Atomic force microscopy (AFM). Gene expression analysis of exosome biogenesis of early and late retinal organoids were compared using qPCR. The protein profile of the EVs were analyzed with proteomic tools. RESULTS: NTA indicated the average size of EV as 100-250 nm. A high expression of exosome biogenesis genes was observed in late retinal organoids EVs. Immunoblot analysis showed highly expressed exosomal markers in late retinal organoids EVs compared to early retinal organoids EVs. Protein profiling of retinal organoid EVs displayed a higher differential expression of retinal function-related proteins and EV biogenesis proteins than hUCMSC EVs, implicating that the use of retinal organoid EVs may have a superior therapeutic effect on retinal disorders. CONCLUSION: This study provides supplementary knowledge on the properties of retinal organoid EVs and suggests their potential use in the diagnostic and therapeutic treatments for ocular diseases.

Retinal Organoids: A Human Model System for Development, Diseases, and Therapies.

Inherited retinal degenerations (IRD) encompasses a group of heterogeneous disorders causing debilitating visual diseases and blindness, affecting more than two million people worldwide, in all age groups. The inheritance patterns vary from autosomal dominant, autosomal recessive, X-linked, and sporadic with mutations in over 260 genes identified to date. Despite the significant advances in clinical diagnosis, there is no effective treatment available. Human-induced pluripotent stem cells (hiPSC) derived in vitro 3D retinal organoids offer a powerful preclinical tool to investigate the molecular mechanism(s) of inherited diseases. Organoids have the potential for the development of personalized therapies by modeling the disease-specific and patient-specific IRD. This mini-review will elaborate on the utility of the advanced culture model system by focusing on staging the in vitro human retinogenesis, modeling retinal diseases, and as a tool for testing potential therapeutic approaches to restore or prevent vision loss in affected individuals.

Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a γ-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by histology and bulk RNA-sequencing. We found that this treatment caused supernumerary cone formation in an overwhelmingly synchronized fashion. This analysis revealed several categorical patterns of gene expression changes over time relative to DMSO treated control explants. These were placed in the temporal context of the activation of Otx2, a transcription factor that is expressed at the onset of photoreceptor development and that is required for both rod and cone formation. One group of interest had genes, such as Mybl1, Ascl1, Neurog2, and Olig2, that became upregulated by DAPT treatment before Otx2. Two other groups showed upregulated gene expression shortly after Otx2, either transiently or permanently. This included genes such as Mybl1, Meis2, and Podxl. Our data provide a developmental timeline of the gene expression events that underlie the initial steps of cone genesis and maturation. Applying this strategy to human retinal organoid cultures was also sufficient to induce a massive increase in cone genesis. Taken together, our results provide a temporal framework that can be used to elucidate the gene regulatory logic controlling cone photoreceptor development.

Immunological Considerations for Retinal Stem Cell Therapy.

There is an increasing effort toward generating replacement cells for neuronal application due to the nonregenerative nature of these tissues. While much progress has been made toward developing methodologies to generate these cells, there have been limited improvements in functional restoration. Some of these are linked to the degenerative and often nonreceptive microenvironment that the new cells need to integrate into. In this chapter, we will focus on the status and role of the immune microenvironment of the retina during homeostasis and disease states. We will review changes in both innate and adaptive immunity as well as the role of immune rejection in stem cell replacement therapies. The chapter will end with a discussion of immune-modulatory strategies that have helped to ameliorate these effects and could potentially improve functional outcome for cell replacement therapies for the eye.

Illustrating the potency of current Good Manufacturing Practice-compliant induced pluripotent stem cell lines as a source of multiple cell lineages using standardized protocols.

BACKGROUND AIMS: We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line RESULTS: Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology CONCLUSIONS: We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines.

DEPTOR is a stemness factor that regulates pluripotency of embryonic stem cells.

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.

Hypoxia induces re-entry of committed cells into pluripotency.

Adult stem cells reside in hypoxic niches, and embryonic stem cells (ESCs) are derived from a low oxygen environment. However, it is not clear whether hypoxia is critical for stem cell fate since for example human ESCs (hESCs) are able to self-renew in atmospheric oxygen concentrations as well. We now show that hypoxia can govern cell fate decisions since hypoxia alone can revert hESC- or iPSC-derived differentiated cells back to a stem cell-like state, as evidenced by re-activation of an Oct4-promoter reporter. Hypoxia-induced "de-differentiated" cells also mimic hESCs in their morphology, long-term self-renewal capacity, genome-wide mRNA and miRNA profiles, Oct4 promoter methylation state, cell surface markers TRA1-60 and SSEA4 expression, and capacity to form teratomas. These data demonstrate that hypoxia can influence cell fate decisions and could elucidate hypoxic niche function.

Human embryonic stem cell applications for retinal degenerations.

Loss of vision in severe retinal degenerations often is a result of photoreceptor cell or retinal pigment epithelial cell death or dysfunction. Cell replacement therapy has the potential to restore useful vision for these individuals especially after they have lost most or all of their light-sensing cells in the eye. A reliable, well-characterized source of retinal cells will be needed for replacement purposes. Human embryonic stem cells (ES cells) can provide an unlimited source of replacement retinal cells to take over the function of lost cells in the eye. The author's intent for this review is to provide an historical overview of the field of embryonic stem cells with relation to the retina. The review will provide a quick primer on key pathways involved in the development of the neural retina and RPE followed by a discussion of the various protocols out in the literature for generating these cells from non-human and human embryonic stem cells and end with in vivo application of ES cell-derived photoreceptors and RPE cells.

Disease modeling and pharmacological rescue of autosomal dominant retinitis pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4 months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

Blimp1 controls photoreceptor versus bipolar cell fate choice during retinal development.

Photoreceptors, rods and cones are the most abundant cell type in the mammalian retina. However, the molecules that control their development are not fully understood. In studies of photoreceptor fate determination, we found that Blimp1 (Prdm1) is expressed transiently in developing photoreceptors. We analyzed the function of Blimp1 in the mouse retina using a conditional deletion approach. Developmental analysis of mutants showed that Otx2(+) photoreceptor precursors ectopically express the bipolar cell markers Chx10 (Vsx2) and Vsx1, adopting bipolar instead of photoreceptor fate. However, this fate shift did not occur until the time when bipolar cells are normally specified during development. Most of the excess bipolar cells died around the time of bipolar cell maturation. Our results suggest that Blimp1 expression stabilizes immature photoreceptors by preventing bipolar cell induction. We conclude that Blimp1 regulates the decision between photoreceptor and bipolar cell fates in the Otx2(+) cell population during retinal development.

Factors Affecting Stem Cell-Based Regenerative Approaches in Retinal Degeneration.

Inherited and age-associated vision loss is often associated with degeneration of the cells of the retina, the light-sensitive layer at the back of the eye. The mammalian retina, being a postmitotic neural tissue, does not have the capacity to repair itself through endogenous regeneration. There has been considerable excitement for the development of cell replacement approaches since the isolation and development of culture methods for human pluripotent stem cells, as well as the generation of induced pluripotent stem cells. This has now been combined with novel three-dimensional organoid culture systems that closely mimic human retinal development in vitro. In this review, we cover the current state of the field, with emphasis on the cell delivery challenges, role of the recipient immunological microenvironment, and challenges related to connectivity between transplanted cells and host circuitry both locally and centrally to the different areas of the brain.

Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases.

One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.

Disease modeling and pharmacological rescue of autosomal dominant Retinitis Pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

The importance of unambiguous cell origin determination in neuronal repopulation studies.

Neuronal repopulation achieved through transplantation or transdifferentiation from endogenous sources holds tremendous potential for restoring function in chronic neurodegenerative disease or acute injury. Key to the evaluation of neuronal engraftment is the definitive discrimination of new or donor neurons from preexisting cells within the host tissue. Recent work has identified mechanisms by which genetically encoded donor cell reporters can be transferred to host neurons through intercellular material transfer. In addition, labeling transplanted and endogenously transdifferentiated neurons through viral vector transduction can yield misexpression in host cells in some circumstances. These issues can confound the tracking and evaluation of repopulated neurons in regenerative experimental paradigms. Using the retina as an example, we discuss common reasons for artifactual labeling of endogenous host neurons with donor cell reporters and suggest strategies to prevent erroneous conclusions based on misidentification of cell origin.

Characterization of Retinal Development in 13-Lined Ground Squirrels.

Purpose: The cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human central retina, but a thorough examination of retinal development in this species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize 13-LGS as a practical alternative animal model for investigating cone-based vision in health and disease. Methods: The spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Postnatal gene expression changes were validated by quantitative PCR. Results: The 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell-type-specific marker labeling was at embryonic stage 18 (E18) with ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E22.5 with photoreceptor progenitors (Otx2, recoverin) followed shortly by horizontal and amacrine cells (Lhx1, Oc1) at E24 to E25.5; and at postnatal stage 15 (P15) with bipolar cells (Vsx1, CaBP5) and Müller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin-positive outer segments and peanut agglutinin (PNA) labeling of cone sheaths was completed at the time of eye opening (P21-P24). Conclusions: The timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. Translational Relevance: This thorough examination of an emerging translationally relevant cone-dominant specie provides a baseline for future disease modeling and stem cell approach studies of human vision.

Timed Notch Inhibition Drives Photoreceptor Fate Specification in Human Retinal Organoids.

Purpose: Transplanting photoreceptors from human pluripotent stem cell-derived retinal organoids have the potential to reverse vision loss in affected individuals. However, transplantable photoreceptors are only a subset of all cells in the organoids. Hence, the goal of our current study was to accelerate and synchronize photoreceptor differentiation in retinal organoids by inhibiting the Notch signaling pathway at different developmental time-points using a small molecule, PF-03084014 (PF). Methods: Human induced pluripotent stem cell- and human embryonic stem cells-derived retinal organoids were treated with 10 µM PF for 3 days starting at day 45 (D45), D60, D90, and D120 of differentiation. Organoids were collected at post-treatment days 14, 28, and 42 and analyzed for progenitor and photoreceptor markers and Notch pathway inhibition by immunohistochemistry (IHC), quantitative PCR, and bulk RNA sequencing (n = 3-5 organoids from three independent experiments). Results: Retinal organoids collected after treatment showed a decrease in progenitor markers (KI67, VSX2, PAX6, and LHX2) and an increase in differentiated pan-photoreceptor markers (OTX2, CRX, and RCVRN) at all organoid stages except D120. PF-treated organoids at D45 and D60 exhibited an increase in cone photoreceptor markers (RXRG and ARR3). PF treatment at D90 revealed an increase in cone and rod photoreceptors markers (ARR3, NRL, and NR2E3). Bulk RNA sequencing analysis mirrored the immunohistochemistry data and quantitative PCR confirmed Notch effector inhibition. Conclusions: Timing the Notch pathway inhibition in human retinal organoids to align with progenitor competency stages can yield an enriched population of early cone or rod photoreceptors.

A method for stabilizing RNA for transfection that allows control of expression duration.

RNA transfection methods have not proven to be as popular as DNA methods due to the highly transient nature of the RNA inside the cell. However, there are many advantages in using RNA for gene over-expression, such as the rapidity of expression, the ability to express in all cell types without the need for cell-type-specific promoters, and the ability to analyze the effects of gene over-expression in a transient manner. Therefore, we have developed a method (StabiLizingUtr: SLU) to stabilize the RNA for varying durations, using specific sequences from the 3'UTR of the Venezuelan equine encephalitis virus (VEEV). We have designed a plasmid for cloning genes upstream from repeated stabilizing sequences to generate mRNA with one or more VEEV-stabilizing sequence motifs. We demonstrate this method in several cell and tissue types, including the mammalian cochlea, a tissue that has been difficult to transfect with other methods.

Dpp/TGFß-superfamily play a dual conserved role in mediating the damage response in the retina.

Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. Here we identify Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFß) superfamily, that includes TGFß and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, we show that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, we find a dynamic response of retinal cells to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFß/BMP signaling, as supplementation of BMP4 or inhibition of TGFß using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. Our data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained.