Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms.

Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.

Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types.

The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.

Pax6 organizes the anterior eye segment by guiding two distinct neural crest waves.

Cranial neural crest (NC) contributes to the developing vertebrate eye. By multidimensional, quantitative imaging, we traced the origin of the ocular NC cells to two distinct NC populations that differ in the maintenance of sox10 expression, Wnt signalling, origin, route, mode and destination of migration. The first NC population migrates to the proximal and the second NC cell group populates the distal (anterior) part of the eye. By analysing zebrafish pax6a/b compound mutants presenting anterior segment dysgenesis, we demonstrate that Pax6a/b guide the two NC populations to distinct proximodistal locations. We further provide evidence that the lens whose formation is pax6a/b-dependent and lens-derived TGFß signals contribute to the building of the anterior segment. Taken together, our results reveal multiple roles of Pax6a/b in the control of NC cells during development of the anterior segment.

Stanniocalcin-1 Is an Ocular Hypotensive Agent and a Downstream Effector Molecule That Is Necessary for the Intraocular Pressure-Lowering Effects of Latanoprost.

Purpose: To identify downstream signaling molecules through which intraocular pressure (IOP) is lowered following treatment with the prostaglandin analog latanoprost. Methods: Total RNA and protein isolated from primary human Schlemm's canal cells (n = 3) treated with latanoprost (free acid; 100 nM) were processed for quantitative PCR and Western blot analysis. IOP was evaluated in stanniocalcin-1 (STC-1-/-) and wild-type mice following treatment with latanoprost or Rho kinase inhibitor Y27632. Human anterior segment pairs (n = 8) were treated with recombinant STC-1 (5, 50, or 500 ng/mL) and pressure was recorded using custom-designed software. The effect of recombinant STC-1 (0.5 mg/mL) on IOP was evaluated in wild-type mice. Tissue morphology was evaluated by light and transmission electron microscopy. Results: Increased STC-1 mRNA (4.0- to 25.2-fold) and protein expression (1.9- to 5.1-fold) was observed within 12 hours following latanoprost treatment. Latanoprost reduced IOP in wild-type mice (22.0% ± 1.9%), but had no effect on STC-1-/- mice (0.5% ± 0.7%). In contrast, Y27632 reduced IOP in both wild-type (12.5% ± 1.2%) and in STC-1-/- mice (13.1% ± 2.8%). Human anterior segments treated with STC-1 (500 ng/mL) showed an increase in outflow facility (0.15 ± 0.03 to 0.27 ± 0.09 µL/min/mm Hg) while no change was observed in paired vehicle-treated controls. Recombinant STC-1 reduced IOP in wild-type mice by 15.2% ± 3.0%. No observable morphologic changes were identified between treatment groups when evaluated by microscopy. Conclusions: Latanoprost-induced reduction of IOP is mediated through the downstream signaling molecule STC-1. When used by itself, STC-1 exhibits ocular hypotensive properties.

[Magnetic Resonance Microscopy of the Accommodative Apparatus]. / Magnetresonanzmikroskopie des Akkommodationsapparats.

Magnetic resonance microscopy (MRM) at ultra-high magnetic fields allows acquisition of high resolution MR images in the micrometre range. The use of ultra-high magnetic fields opens the possibility of user-independent and artefact-free detailed characterisation of the anatomical tissue of the human eye, which is not achievable with classical imaging techniques. This article correlates MRM of the anterior eye segment and the accommodative apparatus at 9.4 Tesla with conventional histology.

Tele-manufactured affordable smartphone anterior segment microscope.

The recent advances in mobile technology have made the smartphone a powerful and accessible tool. This article describe the development of a novel smartphone-based anterior segment microscope that is compatible with tele-manufacturing. The anterior segment microscope is equipped with both cobalt-blue and red-free filters that can be used for clinical photo-documentation. The digital files of the microscope are transferrable and compatible with additive-manufacturing. Therefore, the entire device can be locally manufactured with rapid prototyping techniques such as 3D printing.

Species Differences in the Geometry of the Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals.

PURPOSE: To determine the impact of anterior segment geometry on ocular scoring systems quantifying anterior chamber (AC) cells in humans and 7 common laboratory species. METHODS: Using normative anterior segment dimensions and novel geometric formulae, ocular section volumes measured by 3 scoring systems; Standardization of Uveitis Nomenclature (SUN), Ocular Services On Demand (OSOD), and OSOD-modified SUN were calculated for each species, respectively. Calculated volumes were applied to each system's AC cell scoring scheme to determine comparative cell density (cells/mm(3)). Cell density values for all laboratory species were normalized to human values and conversion factors derived to create modified scoring schemes, facilitating interspecies comparison with each system, respectively. RESULTS: Differences in anterior segment geometry resulted in marked differences in optical section volume measured. Volumes were smaller in rodents than dogs and cats, but represented a comparatively larger percentage of AC volume. AC cell density (cells/mm(3)) varied between species. Using the SUN and OSOD-modified SUN systems, values in the pig, dog, and cat underestimated human values; values in rodents overestimated human values. Modified normalized scoring systems presented here account for species-related anterior segment geometry and facilitate both intra- and interspecies analysis, as well as translational comparison. CONCLUSIONS: Employment of modified AC cell scoring systems that account for species-specific differences in anterior segment anatomy would harmonize findings across species and may be more predictive for determining ocular toxicological consequences in ocular drug and device development programs.

Reproducibility of angle metrics using the time-domain anterior segment optical coherence tomography: intra-observer and inter-observer variability.

PURPOSE: To evaluate the reproducibility of anterior chamber angle measurements obtained by the Zeiss Visante anterior segment optical coherence tomography (AS-OCT). METHODS: Twenty eyes from 20 normal subjects with open anterior chamber angles were studied. The anterior chamber angle was imaged using the Visante AS-OCT. The angle-opening distance (AOD 500, AOD 750), trabercular iris space area (TISA 500, TISA 750) and scleral spur angle (SS angle) at the inferior angle location were measured. All the subjects underwent imaging in a darkened room (1 foot candles measured at the eye). Images were graded in a masked fashion by certified Doheny Image Reading Center graders. For intra-grader reproducibility assessments, images were re-graded by the same grader 1 week later after random sorting of images. For inter-grader assessments, a second masked grader independently reviewed the images. Intraclass correlation coefficients (ICC) were used to assess reproducibility. RESULTS: Inferior angle measurements of AOD (500, 750), TISA (500, 750) and SS angle for 20 normal eyes were calculated. The intra-observer ICC calculations showed excellent reproducibility for all measurements (AOD 500 = 0.95, AOD 750 = 0.97, TISA 500 = 0.93, TISA 750 = 0.94, SS = 0.96; p < 0.001 for all). The inter-observer ICC calculations showed lower reproducibility for all measurements (AOD 500 = 0.71, p < 0.001; AOD 750 = 0.82, p < 0.001; TISA 500 = 0.49, p = 0.08; TISA 750 = 0.61, p = 0.02; SS = 0.75). CONCLUSION: Determination of anterior chamber angle measurements was possible with the time-domain AS-OCT, but only modest inter-observer reproducibility was found even among experienced graders.

Dual-channel spectral-domain optical-coherence tomography system based on 3 × 3 fiber coupler for extended imaging range.

We have demonstrated a dual-channel multiplexing spectral-domain optical-coherence tomography (SD-OCT) system based on a 3×3 fiber coupler for extended imaging range of whole human eye depth, with a single light source and spectrometer. OCT images of anterior segments of a human eye were sequentially performed and constructed to demonstrate an extended depth range as large as 15 mm in air. A good quality OCT image of the whole anterior segment of an eye was present. Furthermore, whole eye segmental imaging was performed and ocular distances were calculated to show the validation of the system for whole eye morphological measurement.

Age-related changes in human corneal epithelial thickness measured with anterior segment optical coherence tomography.

PURPOSE: To measure corneal and limbal epithelial thickness (ET) in normal subjects and to evaluate its variation with age by using anterior segment optical coherence tomography (AS-OCT). METHODS: A total of 180 normal subjects (180 healthy eyes) were enrolled and divided into four groups according to age: A (0-20 years), B (21-40 years), C (41-60 years), and D (>60 years). Cornea and limbus were imaged with OCT. Corneal ET (CET) was obtained automatically by the built-in analysis software of the OCT system. Limbal ET (LET) in four quadrants was manually measured from OCT images. RESULTS: Corneal ET of a central 2-mm diameter zone in groups A, B, C, and D were 53.4 ± 2.8 µm, 53.4 ± 2.7 µm, 53.2 ± 3.0 µm, and 52.9 ± 3.3 µm, respectively, N showing no significant change with aging. In the paracentral zone extending to 6-mm diameter, correlation analysis suggested that CET was inversely associated with age (P < 0.05). Limbal ET in the nasal and the temporal quadrants were similar and decreased with aging, the averages were 58.3 ± 8.1 µm, 54.1 ± 6.1 µm, 51.2 ± 6.1 µm, 51.6 ± 5.2 µm for groups A, B, C and D, respectively; while age seemed to have no effect on LET of the superior and the inferior quadrant. CONCLUSIONS: The paracentral corneal epithelium, as well as the nasal and temporal limbal epithelium, became thinner with aging, while the central CET seemed to remain constant. Measurement with AS-OCT of the corneal and limbal ET could aid in clinical assessment and planning treatments of the cornea.

Automatic intraocular lens segmentation and detection in optical coherence tomography images.

We present a new algorithm for automatic segmentation and detection of an accommodative intraocular lens implanted in a biomechanical eye model. We extracted lens curvature and position. The algorithm contains denoising and fan correction by a multi-level calibration routine. The segmentation is realized by an adapted canny edge detection algorithm followed by a detection of lens surface with an automatic region of interest search to suppress non-optical surfaces like the lens haptic. The optical distortion of lens back surface is corrected by inverse raytracing. Lens geometry was extracted by a spherical fit. We implemented and demonstrated a powerful algorithm for automatic segmentation, detection and surface analysis of intraocular lenses in vitro. The achieved accuracy is within the expected range determined by previous studies. Future improvements will include the transfer to clinical anterior segment OCT devices.

A magnetic bead-based method for mouse trabecular meshwork cell isolation.

PURPOSE: Mice have been used widely for glaucoma research. However, due to the small size of the mouse eye, it is difficult to dissect mouse trabecular meshwork (MTM) tissues and establish MTM cell strains. To circumvent this problem, we took advantage of the phagocytic property of trabecular meshwork (TM) cells, and developed a novel magnetic bead-based method that enables us to isolate pure MTM cells. METHODS: After anesthesia, up to 2 µL of fluorescent or magnetic microbeads were injected intracamerally into the mouse eyes. To study the distribution and localization of the beads, mice were sacrificed 1 to 7 days after injection, and eyes were enucleated for fluorescent or transmission electron microscopy (TEM) study, respectively. To isolate MTM cells, anterior segments injected with magnetic beads were dissected from 10 to 15 sterilized mouse eyes 7 days after injection. The tissues were digested with collagenase A and purified by using a magnetic field as well as repeated washing. RESULTS: TEM studies showed that the magnetic beads were located in the mouse TM, but not in corneal or scleral fibroblast cells. Cultured MTM cells were similar morphologically to human TM cells. MTM cells expressed TM markers, including collagen IV, laminin, and α-smooth muscle actin. Also, MTM cells treated with 100 nM dexamethasone showed increased formation of cross-linked actin networks and induction of myocilin expression. CONCLUSIONS: The magnetic bead-based method is efficient for isolating MTM cells with minimal microdissection techniques required. It will be a useful approach for isolating TM cells from small animals for glaucoma research.

A GPR18-based signalling system regulates IOP in murine eye.

BACKGROUND AND PURPOSE: GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N-arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn-CBD). The presence and/or function of any GPR18-based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55. EXPERIMENTAL APPROACH: IOP was measured in mice following topical application of Abn-CBD, NAGly or the GPR55/GPR18 agonist O-1602, alone or with injection of the GPR18 antagonist, O-1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS-MS. KEY RESULTS: GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn-CBD and NAGly, but not O-1602, significantly reduced IOP in all mice tested. The antagonist, O-1918, blocked the effects of Abn-CBD and NAGly. CONCLUSIONS AND IMPLICATIONS: We present evidence for a functional GPR18-based signalling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn-CBD and NAGly, reduce IOP independently of CB1, CB2 or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications.

Simultaneous swept source optical coherence tomography of the anterior segment and retina using coherence revival.

We report on an implementation of coherence revival-based heterodyne swept source optical coherence tomography that is capable of simultaneously imaging the anterior and posterior eye. A polarization-encoded sample arm was used to efficiently focus orthogonal polarizations on the anterior segment and retina. Depth encoding was achieved using coherence revival, which allows for multiple depths within a sample to be simultaneously imaged and frequency encoded by carefully controlling the optical pathlength of each sample path. This design is a significant step toward whole-eye optical coherence tomography (OCT), which would enable customized ray-traced modeling of patient eyes to improve refractive surgical interventions and eliminate optical artifacts in retinal OCT diagnostics. We demonstrated the feasibility of this system for in vivo imaging by simultaneously acquiring images of the anterior segments and retinas in healthy human volunteers.

The effect of Rho-associated protein kinase inhibitor on monkey Schlemm's canal endothelial cells.

PURPOSE: To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells. METHODS: SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632-induced changes in the intracellular localization of ZO-1, claudin-5, ß-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system. RESULTS: Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions. CONCLUSIONS: Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.

Extended in vivo anterior eye-segment imaging with full-range complex spectral domain optical coherence tomography.

We demonstrate the capability of full-range complex (FRC) spectral domain optical coherence tomography (SD-OCT) to image the anterior eye segment from the cornea to the posterior surface of the lens. With an adapted spectrometer design, we developed a SD-OCT system with an extended normal (single half-space) depth range of 7 mm (in air). This OCT-intrinsic depth range was doubled with a FRC technique. We demonstrate the performance of our OCT system by imaging the whole anterior segment of a healthy human eye in vivo.

Tissue discrimination in anterior eye using three optical parameters obtained by polarization sensitive optical coherence tomography.

We developed a tissue discrimination algorithm of polarization sensitive optical coherence tomography (PS-OCT) based on the optical properties of tissues. We calculated the three-dimensional (3D) feature vector from the parameters intensity, extinction coefficient, birefringence, which were obtained by PS-OCT. The tissue type of each pixel was determined according to the position of the feature vector in the 3D feature space. The algorithm was applied for discriminating tissues of the human anterior eye segment. The conjunctiva, sclera, trabecular meshwork (TM), cornea, and uvea were well separated in the 3D feature space, and we observed them with good contrast. The TM line can be observed in the 3D discriminated volume, as observed by gonioscopy.We validated our method by applying our algorithm and histological data to porcine eyes. A marker was injected into sub-Tenon's space and the tissues that were anterior to the marker and posterior to the marker were successfully segmented by our algorithm.

SPARC-null mice exhibit lower intraocular pressures.

PURPOSE: SPARC is a matricellular protein that is highly expressed in remodeling tissues, including the trabecular meshwork and ciliary body. The hypothesis for the study was that SPARC contributes to the regulation of intraocular pressure (IOP). The IOPs of SPARC-null mice, their corresponding wild-type (WT), and heterozygous animals were compared. METHODS: Diurnal and nocturnal IOPs of C57Bl/6x129SvJ WT, SPARC-null, and heterozygous mice were measured. Fluorophotometric measurements were made to assess aqueous turnover. Central corneal thickness (CCT) was measured using histology, ultrasound biomicroscopy, and optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM). RESULTS: During the day, the mean IOP of SPARC-null mice (n = 142, 16.9 +/- 2.4 mm Hg) was lower than that of both WT mice (n = 104, 19.9 +/- 2.9 mm Hg; P < 10(-12)), and heterozygotes (n = 38, 19.3 +/- 2.5 mm Hg; P < 10(-4)). At night, SPARC-null mice also exhibited a blunted increase in IOP in comparison to WT and heterozygous mice. CCTs were not significantly different between WT and SPARC-null mice. Heterozygous mice tended to have thicker corneas (3.4%). Fluorophotometric measurements suggest that aqueous turnover rates in SPARC-null mice are equal to if not greater than rates in WT mice. LM of the SPARC-null iridocorneal angle revealed morphology that is indistinguishable from WT. CONCLUSIONS: SPARC-null mice have lower IOPs than do their WT counterparts with equal CCTs. The rate of aqueous turnover suggests that the mechanism is enhanced outflow resistance.

PD-L1 expression on human ocular cells and its possible role in regulating immune-mediated ocular inflammation.

PURPOSE: To assess the expression of PD-L1 and PD-L2 on human ocular cells and their potential to regulate ocular inflammation. METHODS: Five categories of human ocular cells were evaluated for PD-L1 and PD-L2 expression by RT-PCR and flow cytometry. Three normal eyes and an inflamed eye from a patient with sympathetic ophthalmia were examined by immunohistochemistry for in situ PD-L1 expression. The immunomodulatory functions of PD-L1 and PD-L2 were tested by coculturing untreated or IFN-gamma-pretreated ocular cells with activated human peripheral blood T cells for 48 hours and assessing T-cell production of IFN-gamma, TNF-alpha, IL-4, and IL-5 by ELISA and T-cell apoptosis by flow cytometry. RESULTS: PD-L1 protein was expressed constitutively in 4 of 5 human ocular cell lines, and its expression was significantly upregulated after stimulation by IFN-gamma. Moreover, in situ expression of PD-L1 in inflamed ocular tissues was remarkably upregulated compared with normal eyes. Although PD-L2 expression was detectable by flow cytometry on 3 of 5 ocular cell lines, immunohistochemical staining did not show expression of PD-L2 on either normal or inflamed ocular tissues. IFN-gamma, TNF-alpha, and IL-5 production by activated T cells cocultured with ocular cells was significantly enhanced in the presence of anti-PD-L1 blocking antibody. However, ocular cell-expressed PD-L1 and PD-L2 did not induce T-cell apoptosis. CONCLUSIONS: PD-L1 expressed on human ocular cells has a presumptive role in controlling ocular inflammation by inhibiting the production of proinflammatory cytokines and a Th2 cytokine by activated T cells. This may represent an important mechanism for maintaining immune privilege in the eye.

Decellularizing corneal stroma using N2 gas.

PURPOSE: To examine the efficacy of a novel method of decellularizing porcine corneal stroma using N(2) gas from liquid N(2) and the feasibility of using decellularized porcine corneal stroma in a corneal transplantation model in rabbits. METHODS: Porcine corneas were placed in a tube, and N(2) gas from liquid N(2) was poured into the tube to freeze the corneas and make the inside of the tube hypoxic. After fastening the cap firmly, the tube was kept at room temperature for seven days, and the porcine corneas were examined histologically. A porcine corneal stromal disk treated with the aforementioned method was inserted into a pocket of rabbit corneal stroma and observed for six months. RESULTS: Hoechst 33342 and hematoxylin and eosin staining both showed few cellular nuclei in the porcine corneal stroma incubated in N(2) gas for one week. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay showed many positively stained nuclei in the porcine corneal stroma incubated in N(2) gas for three days. The porcine corneal stroma that was decellularized and transplanted into a rabbit corneal stromal pocket remained clear for six months after transplantation. CONCLUSIONS: This method using N(2) gas decellularizes corneal stroma without reducing corneal transparency.