Bipolar Cells Research Articles

The Voltage-Gated Sodium Channel in Drosophila, Para, Localizes to Dendrites As Well As Axons in Mechanosensitive Chordotonal Neurons.

The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). Here, we show that, in addition to TRP channels, the sole voltage-gated sodium channel (NaV) in Drosophila, Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 μm from the soma in multipolar neurons and 20-40 μm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston's organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable us to better understand Para's role in mechanosensitive transduction.

Nonselective Expression of Short-Wavelength Cone Opsin Improves Learning in Mice with Retinal Degeneration in a Visually Guided Task.

The study explored the potential of an animal opsin nonselectively expressed in various neuronal elements of the degenerative retina to restore the impaired visual function. A knockout murine model of inherited retinal dystrophy was used. Mice were injected intravitreally with either a virus carrying the gene of short-wavelength cone opsin associated with a reporter fluorescent protein or a control virus carrying the sequence of a modified fluorescent protein with enhanced membrane tropism. Viral transduction induced pronounced opsin expression in ganglion, bipolar, and horizontal retinal neurons. Behavioral testing included the visually guided task in the trapezoid Morris water maze and showed a partial recovery of the learning ability in the mice whose retinas had been transduced with cone opsin.

Coupled electron transfer reactions in closed bipolar cells: The impact of asymmetric mass transport

Coupled electron transfer reactions in closed bipolar cells: The impact of asymmetric mass transport

Mutation of vsx genes in zebrafish highlights the robustness of the retinal specification network.

Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors' specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR/Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted toward photoreceptor or Müller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species.

Changes in Choroidal Thickness and Retinal Activity with a Myopia Control Contact Lens.

The axial elongation in myopia is associated with some structural and functional retinal changes. The purpose of this study was to investigate the effect of a contact lens (CL) intended for myopia control on the choroidal thickness (ChT) and the retinal electrical response. Ten myopic eyes (10 subjects, 18-35 years of age) with spherical equivalents from -0.75 to -6.00 diopters (D) were enrolled. The ChT at different eccentricities (3 mm temporal, 1.5 mm temporal, sub-foveal ChT, 1.5 mm nasal, and 3 mm nasal), the photopic 3.0 b-wave of ffERG and the PERG were recorded and compared with two material-matched contact lenses following 30 min of wear: a single-vision CL (SV) and a radial power gradient CL with +1.50 D addition (PG). Compared with the SV, the PG increased the ChT at all eccentricities, with statistically significant differences at 3.0 mm temporal (10.30 ± 11.51 µm, p = 0.020), in sub-foveal ChT (17.00 ± 20.01 µm, p = 0.025), and at 1.5 mm nasal (10.70 ± 14.50 µm, p = 0.044). The PG decreased significantly the SV amplitude of the ffERG photopic b-wave (11.80 (30.55) µV, p = 0.047), N35-P50 (0.90 (0.96) µV, p = 0.017), and P50-N95 (0.46 (2.50) µV, p = 0.047). The amplitude of the a-wave was negatively correlated with the ChT at 3.0T (r = -0.606, p = 0.038) and 1.5T (r = -0.748, p = 0.013), and the amplitude of the b-wave showed a negative correlation with the ChT at 1.5T (r = -0.693, p = 0.026). The PG increased the ChT in a similar magnitude observed in previous studies. These CLs attenuated the amplitude of the retinal response, possibly due to the combined effect of the induced peripheral defocus high-order aberrations impacting the central retinal image. The decrease in the response of bipolar and ganglion cells suggests a potential retrograde feedback signaling effect from the inner to outer retinal layers observed in previous studies.

LIGHT ADAPTATION OF RETINAL ROD BIPOLAR CELLS.

The sensitivity of retinal cells is altered in background light to optimize the detection of contrast. For scotopic (rod) vision, substantial adaptation occurs in the first two cells, the rods and rod bipolar cells (RBCs), through sensitivity adjustments in rods and postsynaptic modulation of the transduction cascade in RBCs. To study the mechanisms mediating these components of adaptation, we made whole-cell, voltage-clamp recordings from retinal slices of mice from both sexes. Adaptation was assessed by fitting the Hill equation to response-intensity relationships with the parameters of half-maximal response (I1/2 ), Hill coefficient (n), and maximum response amplitude (Rmax ). We show that rod sensitivity decreases in backgrounds according to the Weber-Fechner relation with an I1/2 of about 50 R* s-1 The sensitivity of RBCs follows a near-identical function, indicating that changes in RBC sensitivity in backgrounds bright enough to adapt the rods are mostly derived from the rods themselves. Backgrounds too dim to adapt the rods can however alter n, relieving a synaptic nonlinearity likely through entry of Ca2+ into the RBCs. There is also a surprising decrease of Rmax , indicating that a step in RBC synaptic transduction is desensitized or that the transduction channels became reluctant to open. This effect is greatly reduced after dialysis of BAPTA at a membrane potential of +50 mV to impede Ca2+ entry. Thus the effects of background illumination in RBCs are in part the result of processes intrinsic to the photoreceptors and in part derive from additional Ca2+-dependent processes at the first synapse of vision.SIGNIFICANCE STATEMENT:Light adaptation adjusts the sensitivity of vision as ambient illumination changes. Adaptation for scotopic (rod) vision is known to occur partly in the rods and partly in the rest of the retina from presynaptic and postsynaptic mechanisms. We recorded light responses of rods and rod bipolar cells to identify different components of adaptation and study their mechanisms. We show that bipolar-cell sensitivity largely follows adaptation of the rods, but that light too dim to adapt the rods produces a linearization of the bipolar-cell response and a surprising decrease in maximum response amplitude, both mediated by a change in intracellular Ca2+ These findings provide a new understanding of how the retina responds to changing illumination.

Retinal ganglion cells undergo cell type-specific functional changes in a computational model of cone-mediated retinal degeneration.

Understanding the retina in health and disease is a key issue for neuroscience and neuroengineering applications such as retinal prostheses. During degeneration, the retinal network undergoes complex and multi-stage neuroanatomical alterations, which drastically impact the retinal ganglion cell (RGC) response and are of clinical importance. Here we present a biophysically detailed in silico model of the cone pathway in the retina that simulates the network-level response to both light and electrical stimulation. The model included 11, 138 cells belonging to nine different cell types (cone photoreceptors, horizontal cells, ON/OFF bipolar cells, ON/OFF amacrine cells, and ON/OFF ganglion cells) confined to a 300 × 300 × 210μm patch of the parafoveal retina. After verifying that the model reproduced seminal findings about the light response of retinal ganglion cells (RGCs), we systematically introduced anatomical and neurophysiological changes (e.g., reduced light sensitivity of photoreceptor, cell death, cell migration) to the network and studied their effect on network activity. The model was not only able to reproduce common findings about RGC activity in the degenerated retina, such as hyperactivity and increased electrical thresholds, but also offers testable predictions about the underlying neuroanatomical mechanisms. Overall, our findings demonstrate how biophysical changes typified by cone-mediated retinal degeneration may impact retinal responses to light and electrical stimulation. These insights may further our understanding of retinal processing and inform the design of retinal prostheses.

Design of NiNC single atom catalyst layers and AEM electrolyzers for stable and efficient CO2-to-CO electrolysis: Correlating ionomer and cell performance

Deploying single-site NiNC catalysts in cathode catalyst layers of bipolar electrolyzer cells enables catalytic CO2 valorization to e-CO at industrially relevant yields and efficiencies. The performance of the cathode layers is controlled by the turnover frequency of the active sites as well as mass transfer to and from the active sites. While the atomic scale structure-reactivity relations of single-site NiNC catalysts have been extensively studied, the mass transfer characteristics of single atom catalyst layers were poorly discussed. In this work, we design, build, and test NiNC catalyst layers using a novel set of distinct ion exchange ionomer materials and correlate the performance of cathode catalyst layers with their reactivity and stability in full single MEA electrolyzer cells. The Sustainion anion exchange ionomer delivered optimal performance, yielding about 90% CO faradaic efficiency up to 300 mA cm−2 and 15 h stable performance at 200 mA cm−2. Our analysis attributes its favorable electrolyzer performance to its balanced conductivity and hydrophobicity, which mitigates electrode flooding while ensuring excellent ion and CO2 transfer rates even at high current densities.

Ultrathin single-ion conducting polymer enabling a stable Li|Li1.3Al0.3Ti1.7(PO4)3 interface

NASICON-type Li1+xAlxTi2−x(PO4)3 (LATP) solid electrolytes have attracted great attention because of their high ionic conductivity, wide electrochemical stability window, pronounced chemical resistance, and low cost. However, the chemical instability of LATP against metallic lithium (Li0) poses a major challenge and hinders its application in solid-state lithium batteries. Herein, an ultrathin polysiloxane-based single-ion conductor (PSiO) serves as multifunctional protection interlayer to enhance the interfacial stability between LATP and Li0. PSiO effectively blocks the direct contact between Li0 and LATP, regulates the homogeneous Li+ flux at the Li|electrolyte interface, promotes the intimate contact between PSiO and Li0 by forming Si − O − Li bonds, and generates an LiF-enriched Li|electrolyte interphase. As a result, it enables more than 2,000 h of stable cycling in symmetric PSiO@Li‖PSiO@Li cells and superior rate capability and cycling stability in high-energy PSiO@Li‖LiNi0.88Co0.09Mn0.03O2 cells. The realization of well performing 2-layer bipolar stacked cells eventually demonstrates the great potential of this approach.

Molecular docking and In-Ovo analysis of human amniotic fluid extracellular vesicles loaded with sulforaphane: A potential therapy for neurological disorders

Sulforaphane (SFN) is a bioactive component found in broccoli that has neuroprotective and anti-cancer effects. SFN has been encapsulated in various drug carriers to improve its stability and effectiveness. Specifically, this study presents an SFN delivery system based on human Amniotic Fluid-EVs (hAFEVs) for the treatment of neurological disorders. The binding affinity between hAFEVs (<200 nm) and SFN was investigated by spectroscopic analysis, revealing a temperature-dependent binding mechanism. The binding constants (ksv, kq, and kb) were also assessed, indicating a static quenching mechanism. Additionally, the docking studies of SFN and CD9, CD81, HSP70, and TSG101 were performed using AutoDock 4.2.6 software's Lamarckian genetics algorithm to investigate the type of interaction and their effects on the targetability of hAFEVs. The obtained results showed that SFN-loaded hAFEVs had a favorable effect on increasing the survival and proliferation of human PC12 cells (137%). The results of gene expression studies showed that this release system can upregulate the expression of Nrf2 and decrease the expression of IL-6, which shows the support of nerve cells against oxidative stress. In the In-Ovo study, treatment with SFN-hAFEVs resulted in improved grey and white matter cohesion and the improved orientation of bipolar neurons. The results indicate a significant increase in synaptophysin expression and the number of doublecortin-positive cells improved neuronal development and synapse formation. The findings of this study suggest that SFN-hAFEVs have great potential as a treatment for neurological disorders.

Retinal alpha-synuclein accumulation correlates with retinal dysfunction and structural thinning in the A53T mouse model of Parkinson's disease.

Abnormal alpha-synuclein (α-SYN) protein deposition has long been recognized as one of the pathological hallmarks of Parkinson's disease's (PD). This study considers the potential utility of PD retinal biomarkers by investigating retinal changes in a well characterized PD model of α-SYN overexpression and how these correspond to the presence of retinal α-SYN. Transgenic A53T homozygous (HOM) mice overexpressing human α-SYN and wildtype (WT) control littermates were assessed at 4, 6, and 14 months of age (male and female, n = 15-29 per group). In vivo retinal function (electroretinography, ERG) and structure (optical coherence tomography, OCT) were recorded, and retinal immunohistochemistry and western blot assays were performed to examine retinal α-SYN and tyrosine hydroxylase. Compared to WT controls, A53T mice exhibited reduced light-adapted (cone photoreceptor and bipolar cell amplitude, p < 0.0001) ERG responses and outer retinal thinning (outer plexiform layer, outer nuclear layer, p < 0.0001) which correlated with elevated levels of α-SYN. These retinal signatures provide a high throughput means to study α-SYN induced neurodegeneration and may be useful in vivo endpoints for PD drug discovery.

Bipolar cells containing protein kinase Cα mediate attentional facilitation of the avian retinal ganglion cells by the retinopetal signal.

Birds have a well-developed retinopetal system projecting from the midbrain to the contralateral retina. The signal sent to the retina through the retinopetal system facilitates visual responses of the retinal ganglion cells (RGCs), and the retinopetal signals function as attentional signals during visual search. Thus, the retinopetal signal somehow reaches and facilitates visual responses of the RGCs. However, the tertiary neuron of the retinopetal system, the isthmo-optic target cell (IOTC), is unlikely to contact most RGCs directly, because the IOTCs form axon terminals localized in the outermost sublayer (lamina 1) of the inner plexiform layer (IPL) where few RGC dendrites terminate. Therefore, some other intrinsic retinal neurons must be involved in the centrifugal attentional enhancement of visual responses of the RGCs. We investigated connections of the target cells of the IOTCs in chicken and quail, using light and electron microscopic immunohistochemistry. We show that axon terminals of the IOTC make synaptic contacts with protein kinase Cα (PKCα)-immunoreactive (ir) bipolar cells (PKCα-BCs) in lamina 1 of the IPL. Furthermore, with prolonged electrical stimulation of the isthmo-optic nucleus (ION) on one side, whose neurons send their axons to the contralateral retina and make synaptic contacts there with IOTCs, phosphorylation of cAMP response element-binding protein was observed in the PKCα-BCs in the contralateral retina, but not in the ipsilateral retina. This suggests that electrical stimulation of ION activated PKCα-BCs through synapses from IOTCs to PKCα-BCs, thus stimulating transcription in PKCα-BCs. Thus, centrifugal attentional signals may facilitate visual responses of RGCs via the PKCα-BCs.

Direct neuronal reprogramming by temporal identity factors

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors and shift cell fate output, but whether they can also reprogram the identity of terminally differentiated cells is unknown. To address this question, we designed a conditional gene expression system that allows rapid screening of potential reprogramming factors in mouse retinal glial cells combined with genetic lineage tracing. Using this assay, we found that coexpression of the early temporal identity transcription factors Ikzf1 and Ikzf4 is sufficient to directly convert Müller glial (MG) cells into cells that translocate to the outer nuclear layer (ONL), where photoreceptor cells normally reside. We name these "induced ONL (iONL)" cells. Using genetic lineage tracing, histological, immunohistochemical, and single-cell transcriptome and multiome analyses, we show that expression of Ikzf1/4 in MG invivo, without retinal injury, mostly generates iONL cells that share molecular characteristics with bipolar cells, although a fraction of them stain for Rxrg, a cone photoreceptor marker. Furthermore, we show that coexpression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons in culture by rapidly remodeling chromatin and activating a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in terminally differentiated cells.

NON-SELECTIVE EXPRESSION OF SHORT-WAVELENGTH CONE OPSIN IMPROVES LEARNING IN MICE WITH RETINAL DEGENERATION IN A VISUALLY GUIDED TASK

Here, we explore the potential of an animal opsin non-selectively expressed in various neuronal elements of the degenerative retina to restore impaired visual function. In this study, a knockout murine model of inherited retinal distrophy was used. Animals were injected intravitreally with either a virus carrying gene of the short-wavelength cone opsin associated with a reporter fluorescent protein, or a control virus carrying the sequence of a modified fluorescent protein that had an enhanced membrane tropism. The viral transduction induced pronounced opsin expression in ganglion, bipolar, and horizontal retinal neurons. Behavioral testing of the treated mice in the visually guided task in the trapezoid Morris water maze showed a partial recovery of learning ability in animals whose retinas had been transduced with the cone opsin.

A Novel Role for UNC119 as an Enhancer of Synaptic Transmission.

Mammalian UNC119 is a ciliary trafficking chaperone highly expressed in the inner segment of retinal photoreceptors. Previous research has shown that UNC119 can bind to transducin, the synaptic ribbon protein RIBEYE, and the calcium-binding protein CaBP4, suggesting that UNC119 may have a role in synaptic transmission. We made patch-clamp recordings from retinal slices in mice with the UNC119 gene deleted and showed that removal of even one gene of UNC119 has no effect on the rod outer segment photocurrent, but acted on bipolar cells much like background light: it depolarized membrane potential, decreased sensitivity, accelerated response decay, and decreased the Hill coefficient of the response-intensity relationship. Similar effects were seen on rod bipolar-cell current and voltage responses, and after exposure to bright light to translocate transducin into the rod inner segment. These findings indicate that UNC119 deletion reduces the steady-state glutamate release rate at rod synapses, though no change in the voltage dependence of the synaptic Ca current was detected. We conclude that UNC119, either by itself or together with transducin, can facilitate the release of glutamate at rod synapses, probably by some interaction with RIBEYE or other synaptic proteins rather than by binding to CaBP4 or calcium channels.

Assessment of the uniform field electroretinogram for mouse retinal ganglion cell functional analysis.

The uniform field electroretinogram (UF-ERG) has been suggested as an alternative to the pattern electroretinogram (PERG) for non-invasive assessment of retinal ganglion cell (RGC) function in primates. We evaluated the validity of the UF-ERG to assess mouse RGC activity in vivo. Unilateral optic nerve crush (ONC) was performed on adult C57BL/6J mice. Contralateral eyes with uncrushed optic nerves and eyes from surgically naive mice served as experimental controls. Electrophysiological visual assessment was performed at 12weeks post-ONC. Flash-mediated visual-evoked cortical potentials (VEPs) were measured to confirm the robustness of the ONC procedure. Full-field flash ERGs were used to interrogate photoreceptor and retinal bipolar cell function. RGC function was assessed with pattern ERGs. Summed onset and offset UF-ERG responses to alternating dark and light uniform field flash stimuli of different intensities and wavelengths were recorded from ONC and control eyes, and relative differences were compared to the PERG results. Following electrophysiological analysis, RGC loss was monitored by immunohistochemical staining of the RGC marker protein, RBPMS, in post-mortem retinal tissues. ONC dramatically impacts RGC integrity and optic nerve function, demonstrated by reduced RGC counts and near complete elimination of VEPs. ONC did not affect scotopic ERG a-wave and b-wave amplitudes, while PERG amplitudes of eyes subjected to ONC were reduced by approximately 50% compared to controls. Summation of ON and OFF UF-ERG responses did not reveal statistically significant differences between ONC and control eyes, regardless of visual stimulus. PERG responses are markedly impaired upon ONC, while UF-ERG responses are not significantly affected by surgical trauma to RGC axons in mice. The more closely related pattern and uniform field ERGs recorded in primates suggests species-specific differences in RGC features or subpopulations corresponding to PERG and UF-ERG response generators, limiting the utility of the UF-ERG for mouse RGC functional analysis.

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

The histone H3K36 demethylase Fbxl11 plays pivotal roles in the development of retinal late-born cell types.

Histone methylation plays a vital role in retinal development. However, the role of histone H3K36 methylation in retinal development is not clear. We examined the role of H3K36 methylation by loss-of-function analysis of H3K36me1/2 demethylases, Fbxl10, and Fbxl11. We analyzed the effect of knockout of these genes in the developing and mature retina on retinal development. Knockout of Fbxl10 specifically in the developing retina did not result in gross developmental abnormalities. Although adult rod photoreceptor-specific knockout of Fbxl11 in mature retinas did not result in morphological abnormalities, Fbxl11 knockout in developing retinas increased apoptosis, suppressed the proliferation of retinal progenitor cells, and resulted in microphthalmia. Morphological analysis revealed perturbed differentiation of rod photoreceptor and bipolar cells. RNA-seq of retinas at P7 showed markedly decreased expression of genes characterizing rod photoreceptor and bipolar cells in Fbxl11-knockout retinas. In addition, perturbation of alternative splicing increased intron retention in Fbxl11-knockout retinas. Genome-wide evaluation of the H3K36 methylation status revealed that Fbxl11 knockout altered the distribution of H3K36me2/3 in genes important for rod photoreceptor development. Taken together, we show that Fbxl11 plays pivotal roles in the development of retinal late-born cell types and may contribute to tight control of H3K36 methylation during retinal development.

Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.

The light pathways are segregated into rod and cone pathways in which rods synapse with rod bipolar cells (RBCs), while cones contact cone bipolar cells (CBCs). However, previous studies found that cones can make synapse with RBCs (cone-RBC synapses) and rods can contact OFF CBCin primate and rabbit retinas. Recently, such cone-RBC synapses have been reported physiologically and morphologically in the mouse retina. Nevertheless, the precise subcellular evidence to determine whether it is the invaginating synapse or the flat contact remains absent. This is due to a lack of immunochemically verified ultrastructural data. Here, we investigated the precise expression of protein kinase C alpha (PKCα) using pre-embedding immunoelectron microscopy (immuno-EM) with a monoclonal antibody against PKCα, a biomarker for the RBCs. We determined the nanoscale localization of PKCα in the outer plexiform layer of the mouse and guinea pig retinas. Our results demonstrate the existence of both the direct invaginating synapse and the basal/flat contact of the cone-RBCs, providing for the first time immunochemically verified ultrastructural evidence for the cone-RBC synapse in the mouse and guinea pig retinas. These results suggest that the cross talk between cone and rod pathways is much more extensive than previously assumed.

One-stop assembly of adherent 3D retinal organoids from hiPSCs based on 3D-printed derived PDMS microwell platform

The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs), mimicking the growth and development of the human retina, is a promising model for investigating inherited retinal diseases in vitro. However, the efficient generation of homogenous ROs remains a challenge. Here we introduce a novel polydimethylsiloxane (PDMS) microwell platform containing 62 V-bottom micro-cavities for the ROs differentiation from hiPSCs. The uniform adherent 3D ROs could spontaneously form using neural retina (NR) induction. Our results showed that the complex of NR (expressing VSX2), ciliary margin (CM) (expressing RDH10), and retinal pigment epithelium (RPE) (expressing ZO-1, MITF, and RPE65) developed in the PDMS microwell after the differentiation. It is important to note that ROs in PDMS microwell platforms not only enable one-stop assembly but also maintain homogeneity and mature differentiation over a period of more than 25 weeks without the use of BMP4 and Matrigel. Retinal ganglion cells (expressing BRN3a), amacrine cells (expressing AP2a), horizontal cells (expressing PROX1 and AP2α), photoreceptor cells for cone (expressing S-opsin and L/M-opsin) and rod (expressing Rod opsin), bipolar cells (expressing VSX2 and PKCα), and Müller glial cells (expressing GS and Sox9) gradually emerged. Furthermore, we replaced fetal bovine serum with human platelet lysate and established a xeno-free culture workflow that facilitates clinical application. Thus, our PDMS microwell platform for one-stop assembly and long-term culture of ROs using a xeno-free workflow is favorable for retinal disease modeling, drug screening, and manufacturing ROs for clinical translation.