Deciphering the impact of PROM1 alternative splicing on human photoreceptor development and maturation.

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.

A dystroglycan-laminin-integrin axis coordinates cell shape remodeling in the developing Drosophila retina.

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis.

Connexin Expression Is Altered in the Eye Development of Yotari Mice: A Preliminary Study.

This study aimed to explore how Dab1 functional silencing influences the expression patterns of different connexins in the developing yotari (yot) mice eyes as potential determinants of retinogenesis. Using immunofluorescence staining, the protein expression of Dab1, Reelin, and connexin 37, 40, 43, and 45 (Cx37, Cx40, Cx43, and Cx45) in the wild-type (wt) and yot eyes at embryonic days 13.5 and 15.5 (E13.5 and E15.5) were analyzed. Different expression patterns of Cx37 were seen between the wt and yot groups. The highest fluorescence intensity of Cx37 was observed in the yot animals at E15.5. Cx40 had higher expression at the E13.5 when differentiation of retinal layers was still beginning, whereas it decreased at the E15.5 when differentiation was at the advanced stage. Higher expression of Cx43 was found in the yot group at both time points. Cx45 was predominantly expressed at E13.5 in both groups. Our results reveal the altered expression of connexins during retinogenesis in yot mice and their potential involvement in retinal pathology, where they might serve as prospective therapeutic targets.

Proper Frequency of Perinatal Retinal Waves Is Essential for the Precise Wiring of Visual Axons in Nonimage-Forming Nuclei.

The development of the visual system is a complex and multistep process characterized by the precise wiring of retinal ganglion cell (RGC) axon terminals with their corresponding neurons in the visual nuclei of the brain. Upon reaching primary image-forming nuclei (IFN), such as the superior colliculus and the lateral geniculate nucleus, RGC axons undergo extensive arborization that refines over the first few postnatal weeks. The molecular mechanisms driving this activity-dependent remodeling process, which is influenced by waves of spontaneous activity in the developing retina, are still not well understood. In this study, by manipulating the activity of RGCs in mice from either sex and analyzing their transcriptomic profiles before eye-opening, we identified the Type I membrane protein synaptotagmin 13 (Syt13) as involved in spontaneous activity-dependent remodeling. Using these mice, we also explored the impact of spontaneous retinal activity on the development of other RGC recipient targets such as nonimage-forming (NIF) nuclei and demonstrated that proper frequency and duration of retinal waves occurring prior to visual experience are essential for shaping the connectivity of the NIF circuit. Together, these findings contribute to a deeper understanding of the molecular and physiological mechanisms governing activity-dependent axon refinement during the assembly of the visual circuit.

Retinal Input Is Required for the Maintenance of Neuronal Laminae in the Ventrolateral Geniculate Nucleus.

Retinal ganglion cell (RGC) axons provide direct input into several brain regions, including the dorsal lateral geniculate nucleus (dLGN), which is important for image-forming vision, and the ventrolateral geniculate nucleus (vLGN), which is associated with nonimage-forming vision. Through both activity- and morphogen-dependent mechanisms, retinal inputs play important roles in the development of dLGN, including the refinement of retinal projections, morphological development of thalamocortical relay cells (TRCs), timing of corticogeniculate innervation, and recruitment and distribution of inhibitory interneurons. In contrast, little is known about the role of retinal inputs in the development of vLGN. Grossly, vLGN is divided into two domains, the retinorecipient external vLGN (vLGNe) and nonretinorecipient internal vLGN (vLGNi). Studies previously found that vLGNe consists of transcriptionally distinct GABAergic subtypes distributed into at least four adjacent laminae. At present, it remains unclear whether retinal inputs influence the development of these cell-type-specific neuronal laminae in vLGNe. Here, we elucidated the developmental timeline for these laminae in the mouse vLGNe, and results indicate that these laminae are specified at or before birth. We observed that mutant mice without retinal inputs have a normal laminar distribution of GABAergic cells at birth; however, after the first week of postnatal development, these mutants exhibited a dramatic disruption in the laminar organization of inhibitory neurons and clear boundaries between vLGNe and vLGNi. Overall, our results show that while the formation of cell-type-specific layers in mouse vLGNe does not depend on RGC inputs, retinal signals are critical for their maintenance.

Single cell dual-omic atlas of the human developing retina.

The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases.

Single-cell epigenomic reconstruction of developmental trajectories from pluripotency in human neural organoid systems.

Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.

Histological and scanning electron microscope observations on the developing retina of the cuttlefish (Sepia officinalis Linnaeus, 1758).

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis.

Non-canonical Wnt pathway expression in the developing mouse and human retina.

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.

Hyaluronan improves photoreceptor differentiation and maturation in human retinal organoids.

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.

Shedding a Light on Dark Genes: A Comparative Expression Study of PRR12 Orthologues during Zebrafish Development.

Haploinsufficiency of the PRR12 gene is implicated in a human neuro-ocular syndrome. Although identified as a nuclear protein highly expressed in the embryonic mouse brain, PRR12 molecular function remains elusive. This study explores the spatio-temporal expression of zebrafish PRR12 co-orthologs, prr12a and prr12b, as a first step to elucidate their function. In silico analysis reveals high evolutionary conservation in the DNA-interacting domains for both orthologs, with significant syntenic conservation observed for the prr12b locus. In situ hybridization and RT-qPCR analyses on zebrafish embryos and larvae reveal distinct expression patterns: prr12a is expressed early in zygotic development, mainly in the central nervous system, while prr12b expression initiates during gastrulation, localizing later to dopaminergic telencephalic and diencephalic cell clusters. Both transcripts are enriched in the ganglion cell and inner neural layers of the 72 hpf retina, with prr12b widely distributed in the ciliary marginal zone. In the adult brain, prr12a and prr12b are found in the cerebellum, amygdala and ventral telencephalon, which represent the main areas affected in autistic patients. Overall, this study suggests PRR12's potential involvement in eye and brain development, laying the groundwork for further investigations into PRR12-related neurobehavioral disorders.

NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Ocular Necessities: A Neuroethological Perspective on Vertebrate Visual Development.

BACKGROUND: By examining species-specific innate behaviours, neuroethologists have characterized unique neural strategies and specializations from throughout the animal kingdom. Simultaneously, the field of evolutionary developmental biology (informally, "evo-devo") seeks to make inferences about animals' evolutionary histories through careful comparison of developmental processes between species, because evolution is the evolution of development. Yet despite the shared focus on cross-species comparisons, there is surprisingly little crosstalk between these two fields. Insights can be gleaned at the intersection of neuroethology and evo-devo. Every animal develops within an environment, wherein ecological pressures advantage some behaviours and disadvantage others. These pressures are reflected in the neurodevelopmental strategies employed by different animals across taxa. SUMMARY: Vision is a system of particular interest for studying the adaptation of animals to their environments. The visual system enables a wide variety of animals across the vertebrate lineage to interact with their environments, presenting a fantastic opportunity to examine how ecological pressures have shaped animals' behaviours and developmental strategies. Applying a neuroethological lens to the study of visual development, we advance a novel theory that accounts for the evolution of spontaneous retinal waves, an important phenomenon in the development of the visual system, across the vertebrate lineage. KEY MESSAGES: We synthesize literature on spontaneous retinal waves from across the vertebrate lineage. We find that ethological considerations explain some cross-species differences in the dynamics of retinal waves. In zebrafish, retinal waves may be more important for the development of the retina itself, rather than the retinofugal projections. We additionally suggest empirical tests to determine whether Xenopus laevis experiences retinal waves.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.

Integrated Transcriptome Analysis of Long Noncoding RNA and mRNA in Developing and Aging Mouse Retina.

Mice have emerged as a widely employed model for investigating various retinal diseases. However, the availability of comprehensive datasets capturing the entire developmental and aging stages of the mouse retina, particularly during the elderly period, encompassing integrated lncRNA and mRNA expression profiles, is limited. In this study, we assembled a total of 18 retina samples from mice across 6 distinct stages of development and aging (5 days, 3 weeks, 6 weeks, 10 weeks, 6 months, and 15 months) to conduct integrated lncRNA and mRNA sequencing analysis. This invaluable dataset offers a comprehensive transcriptomic resource of mRNA and lncRNA expression profiles during the natural progression of retinal development and aging. The discoveries stemming from this investigation will significantly contribute to the elucidation of the underlying molecular mechanisms associated with various retinal diseases, such as congenital retinal dysplasia and retinal degenerative diseases.

Interommatidial cells build a tensile collagen network during Drosophila retinal morphogenesis.

Drosophila compound eye morphogenesis transforms a simple epithelium into an approximate hollow hemisphere comprised of ∼700 ommatidia, packed as tapering hexagonal prisms between a rigid external array of cuticular lenses and a parallel, rigid internal floor, the fenestrated membrane (FM). Critical to vision, photosensory rhabdomeres are sprung between these two surfaces, grading their length and shape accurately across the eye and aligning them to the optical axis. Using fluorescently tagged collagen and laminin, we show that that the FM assembles sequentially, emerging in the larval eye disc in the wake of the morphogenetic furrow as the original collagen-containing basement membrane (BM) separates from the epithelial floor and is replaced by a new, laminin-rich BM, which advances around axon bundles of newly differentiated photoreceptors as they exit the retina, forming fenestrae in this new, laminin-rich BM. In mid-pupal development, the interommatidial cells (IOCs) autonomously deposit collagen at fenestrae, forming rigid, tension-resisting grommets. In turn, stress fibers assemble in the IOC basal endfeet, where they contact grommets at anchorages mediated by integrin linked kinase (ILK). The hexagonal network of IOC endfeet tiling the retinal floor couples nearest-neighbor grommets into a supracellular tri-axial tension network. Late in pupal development, IOC stress fiber contraction folds pliable BM into a hexagonal grid of collagen-stiffened ridges, concomitantly decreasing the area of convex FM and applying essential morphogenetic longitudinal tension to rapidly growing rhabdomeres. Together, our results reveal an orderly program of sequential assembly and activation of a supramolecular tensile network that governs Drosophila retinal morphogenesis.

Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development.

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.

Astrocyte-intrinsic and -extrinsic Fat1 activities regulate astrocyte development and angiogenesis in the retina.

Angiogenesis is a stepwise process leading to blood vessel formation. In the vertebrate retina, endothelial cells are guided by astrocytes migrating along the inner surface, and the two processes are coupled by a tightly regulated cross-talks between the two cell types. Here, I have investigated how the FAT1 cadherin, a regulator of tissue morphogenesis that governs tissue cross-talk, influences retinal vascular development. Late-onset Fat1 inactivation in the neural lineage in mice, by interfering with astrocyte progenitor migration polarity and maturation, delayed postnatal retinal angiogenesis, leading to persistent vascular abnormalities in adult retinas. Impaired astrocyte migration and polarity were not associated with alterations of retinal ganglion cell axonal trajectories or of the inner limiting membrane. In contrast, inducible Fat1 ablation in postnatal astrocytes was sufficient to alter their migration polarity and proliferation. Altogether, this study uncovers astrocyte-intrinsic and -extrinsic Fat1 activities that influence astrocyte migration polarity, proliferation and maturation, disruption of which impacts retinal vascular development and maintenance.

Emergence of a geometric pattern of cell fates from tissue-scale mechanics in the Drosophila eye.

Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. In this study, we investigate the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. Here, we find that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting, and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven.

Integrated stem cells from apical papilla in a 3D culture system improve human embryonic stem cell derived retinal organoid formation.

AIM: Eye organoids are 3D models of the retina that provide new possibilities for studying retinal development, drug toxicity and the molecular mechanisms of diseases. Although there are several protocols that can be used to generate functional tissues, none have been used to assemble human retinal organoids containing mesenchymal stem cells (MSCs). MAIN METHODS: In this study we intend to assess the effective interactions of MSCs and human embryonic stem cells (hESCs) during retinal organoid formation. We evaluated the inducing activities of bone marrow MSCs (BM-MSCs), trabecular meshwork (TM), and stem cells from apical papilla (SCAP)-derived MSCs in differentiation of hESCs in a three-dimensional (3D) direct co-culture system. KEY FINDINGS: In comparison with the two other MSC sources, the induction potential of SCAP was confirmed in the co-culture system. Although the different SCAP cell ratios did not show any significant morphology changes during the first seven days, increasing the number of SCAPs improved formation of the optic vesicle (OV) structure, which was confirmed by assessment of specific markers. The OVs subsequently developed to an optic cup (OC), which was similar to the in vivo environment. These arrangements expressed MITF in the outer layer and CHX10 in the inner layer. SIGNIFICANCE: We assessed the inducing activity of SCAP during differentiation of hESCs towards a retinal fate in a 3D organoid system. However, future studies be conducted to gather additional details about the development of the eye field, retinal differentiation, and the molecular mechanisms of diseases.