Primary cilia biogenesis and associated retinal ciliopathies.

The primary cilium is a ubiquitous microtubule-based organelle that senses external environment and modulates diverse signaling pathways in different cell types and tissues. The cilium originates from the mother centriole through a complex set of cellular events requiring hundreds of distinct components. Aberrant ciliogenesis or ciliary transport leads to a broad spectrum of clinical entities with overlapping yet highly variable phenotypes, collectively called ciliopathies, which include sensory defects and syndromic disorders with multi-organ pathologies. For efficient light detection, photoreceptors in the retina elaborate a modified cilium known as the outer segment, which is packed with membranous discs enriched for components of the phototransduction machinery. Retinopathy phenotype involves dysfunction and/or degeneration of the light sensing photoreceptors and is highly penetrant in ciliopathies. This review will discuss primary cilia biogenesis and ciliopathies, with a focus on the retina, and the role of CP110-CEP290-CC2D2A network. We will also explore how recent technologies can advance our understanding of cilia biology and discuss new paradigms for developing potential therapies of retinal ciliopathies.

A simple and efficient method for generating human retinal organoids.

Purpose: Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training. Method: We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition. Results: Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium. Conclusions: This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Cell-cell interaction in the pathogenesis of inherited retinal diseases.

The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.

Functional interaction between SNPs and microsatellite in the transcriptional regulation of insulin-like growth factor 1.

A CA-repeat microsatellite in insulin-like growth factor 1 (IGF1) promoter was associated with interindividual variation of circulating IGF1 level. Previously, we reported that such association was due to variation of haplotype unit in a linkage disequilibrium block composed of microsatellite and single-nucleotide polymorphisms (SNPs), suggesting the presence of an interaction between them. In this study, reporter assays were performed to investigate the regulatory effect and interaction of genetic variants on gene expression. We used an in vitro system to compare the transcriptional activities of haplotypes (rs35767:T>C, the CA-repeat microsatellite, rs5742612:T>C, and rs2288377:T>A) in evolutionarily conserved region of IGF1 promoter. In haplotype C-T-T, a longer microsatellite had a lower transcriptional activity (17.6 ± 2.4-fold for 17 repeats and 8.3 ± 1.1-fold for 21 repeats), whereas in haplotype T-C-A, such trend could not be observed, as the microsatellite with 21 repeats had the highest transcriptional activity (17.5 ± 2.3-fold). Because the microsatellite and SNPs affected the transcriptional activity of each other, there may be an interaction between them in the regulation of IGF1 expression. For the first time, we demonstrated that a noncoding microsatellite polymorphism could act as a functional unit and interact with SNPs in the regulation of transcription in human genome.

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects.

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

Leber congenital amaurosis (LCA) is an inherited disease that affects the eyes and causes sight loss in early childhood, which generally gets worse over time. Individuals with this condition have genetic mutations that result in the death of light-sensitive cells, known as photoreceptors, in a region called the retina at the back of the eye. Patients carrying a genetic change in the gene CEP290 account for 20-25% of all LCA. At present, treatment options are only available for a limited number of patients with LCA. One option is to use small molecules as drugs that may target or bypass the faulty processes within the eye to help the photoreceptors survive in many different forms of LCA and other retinal diseases. However, over 90% of new drug candidates fail the first phase of clinical trials for human diseases. This in part due to the candidates having been developed using cell cultures or animal models that do not faithfully reflect how the human body works. Recent advances in cell and developmental biology are now enabling researchers to use stem cells derived from humans to grow retina tissues in a dish in the laboratory. These tissues, known as retinal organoids, behave in a more similar way to retinas in human eyes than those of traditional animal models. However, the methods for making and maintaining human retinal organoids are time-consuming and labor-intensive, which has so far limited their use in the search for new therapies. To address this challenge, Chen et al. developed a large-scale approach to grow retinal organoids from rd16 mutant mice stem cells (which are a good model for LCA caused by mutations to CEP290) and used the photoreceptors from these organoids to screen over 6,000 existing drugs for their ability to promote the survival of photoreceptors. The experiments found that the drug reserpine, which was previously approved to treat high blood pressure, also helped photoreceptors to survive in the diseased organoids. Reserpine also had a similar effect in retinal organoids derived from human patients with LCA and in the rd16 mice themselves. Further experiments suggest that reserpine may help patients with LCA by partially restoring a process by which the body destroys and recycles old and damaged proteins in the cells. The next steps following on from this work will be to perform further tests to demonstrate that this use of reserpine is safe to enter clinical trials as a treatment for LCA and other similar eye diseases.

Nicotinamide Promotes Formation of Retinal Organoids From Human Pluripotent Stem Cells via Enhanced Neural Cell Fate Commitment.

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.

Haplotype effect in the IGF1 promoter accounts for the association between microsatellite and serum IGF1 concentration.

OBJECTIVE: Systemic insulin-like growth factor 1 (IGF1) level is an important risk factor for various diseases. The inter-individual variation of serum IGF1 is determined by environmental and genetic factors, which are attributed to a microsatellite in IGF1 promoter. However, the exact nature of the underlying regulatory elements accounting for this association has not been characterized. Here, we defined the haplotype patterns, including both SNPs and the microsatellite, in the Chinese population, and investigated their regulatory effect on serum IGF1 level. This is the first study in which haplotype patterns of the microsatellite and SNPs in the IGF1 promoter are examined together. METHODS: The linkage disequilibrium (LD) patterns of IGF1 were examined using tagSNPs of the IGF1 regulatory region. The microsatellite, three tagSNPs and haplotypes were correlated with serum IGF1 concentration in 450 normal premenopausal Chinese women. RESULTS: Common alleles of the microsatellite were in strong LD with the three tagSNPs and were associated with particular haplotypes composed of SNPs. Neither the CA repeat number nor SNPs alone showed a robust association with serum IGF1 concentration. On the other hand, the haplotype T-19-A-T was significantly associated with serum IGF1 level. CONCLUSION: No association was found between SNPs and microsatellite alone. However, the haplotype showed better correlation with serum IGF1 level. The results indicate that the previously observed correlation with microsatellite was because of a haplotype effect in the IGF1 promoter. Microsatellite or tagSNPs alone are not the primary regulatory elements of IGF1 expression. The exact regulatory genetic variant needs to be defined by functional genetic studies.

Genetics and therapy for pediatric eye diseases.

Ocular morphogenesis in vertebrates is a highly organized process, orchestrated largely by intrinsic genetic programs that exhibit stringent spatiotemporal control. Alternations in these genetic instructions can lead to hereditary or nonhereditary congenital disorders, a major cause of childhood visual impairment, and contribute to common late-onset blinding diseases. Currently, limited treatment options exist for clinical phenotypes involving eye development. This review summarizes recent advances in our understanding of early-onset ocular disorders and highlights genetic complexities in development and diseases, specifically focusing on coloboma, congenital glaucoma and Leber congenital amaurosis. We also discuss innovative paradigms for potential therapeutic modalities.

HIPRO: A High-Efficiency, Hypoxia-Induced Protocol for Generation of Photoreceptors in Retinal Organoids from Mouse Pluripotent Stem Cells.

Mouse pluripotent stem cells can be efficiently differentiated into retinal organoids with polarized, laminated neural retina harboring all retinal cell types by the Hypoxia-Induced Generation of Photoreceptor in Retinal Organoids (HIPRO) protocol. In our recent publication, we modified the HIPRO protocol on the basis of comparative transcriptome analyses to facilitate photoreceptor biogenesis and maturation. Here, we provide a detailed protocol for efficient generation of retinal organoids from mouse pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to (Chen et al., 2016, DiStefano et al., 2018, Brooks et al., 2019).

Accelerated Development of Rod Photoreceptors in Retinal Organoids Derived from Human Pluripotent Stem Cells by Supplementation with 9-cis Retinal.

Human pluripotent stem cells (PSCs) can be differentiated into retinal organoids with proper neural layer organization, yet the protocols are technically challenging and time consuming. We have modified a widely used differentiation protocol by switching all-trans retinoic acid with 9-cis retinal to accelerate photoreceptor differentiation and improve morphogenesis. In this report, we provide a detailed and improved protocol to generate retinal organoids from human pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to Kaya et al. (2019).

Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development In Vivo.

Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

The mechanism of transactivation regulation due to polymorphic short tandem repeats (STRs) using IGF1 promoter as a model.

Functional short tandem repeats (STR) are polymorphic in the population, and the number of repeats regulates the expression of nearby genes (known as expression STR, eSTR). STR in IGF1 promoter has been extensively studied for its association with IGF1 concentration in blood and various clinical traits and represents an important eSTR. We previously used an in-vitro luciferase reporter model to examine the interaction between STRs and SNPs in IGF1 promoter. Here, we further explored the mechanism how the number of repeats of the STR regulates gene transcription. An inverse correlation between the number of repeats and the extent of transactivation was found in a haplotype consisting of three promoter SNPs (C-STR-T-T). We showed that these adjacent SNPs located outside the STR were required for the STR to function as eSTR. The C allele of rs35767 provides a binding site for CCAAT/enhancer-binding-protein δ (C/EBPD), which is essential for the gradational transactivation property of eSTR and FOXA3 may also be involved. Therefore, we propose a mechanism in which the gradational transactivation by the eSTR is caused by the interaction of one or more transcriptional complexes located outside the STR, rather than by direct binding to a repeat motif of the STR.