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AIM: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). METHODS: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy (TEM) was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and evaluated by flow cytometry, reactive oxygen species (ROS) levels were evaluated by flow cytometry, and adenosine triphosphate (ATP) levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed. RESULTS: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers (Pax6, MITF, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hEROs-RPE and SD-RPE cells. The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80. However, the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80, with hEROs-RPE mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). CONCLUSION: hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria. From the mitochondrial perspective, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.

Summary Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit+/SSEA1− (C-Kit+) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit+ RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment., Highlights •C-Kit+ RPCs migrate and integrate in 3 major layers of advanced degenerative retina • Host inputs evoked by light activates graft neural response • 2-photon imaging visually defines graft-to-host synaptic Ca2+ transients in retina • Grafts connecting with the host retinal circuits preserves vision, In this article Liu, Yin, and colleagues show C-Kit+ retinal progenitor cells can migrate and integrate in three major layers of advanced degenerative retina, form functional synapses with the hosts, and connect with the neural circuits. They provide state-of-the-art techniques to visually demonstrate exceptional synaptic repair and vision restoration, opening up a feasible avenue for advanced RD treatment.

Purpose: Leber congenital amaurosis (LCA) is one of the earliest inherited retinal dystrophies (IRD) that leads to blindness. To date, there have been 25 LCA-associated genes reported in China as well as other countries. The current study aimed to present the dominant molecular genetics and clinical features of LCA in the Han population of western China.Methods: Our study comprised 37 patients with strictly defined Leber congenital amaurosis in a cohort of IRD (2009-2019). The mutations were detected by targeted next-generation sequencing (NGS), Sanger sequencing, and segregation analysis. The patients underwent comprehensive clinical examinations, analysis of phenotypes and genotypes.Results: Out of the 37 patients, 34 harbored known LCA genes; the detection rate of mutations was 91.9%. Forty-seven different alleles incorporated 21 novel mutations; 8 were known LCA-associated genes. The three most frequently mutated genes included CRB1 (27.0%), RDH12 (24.3%), and RPGRIP1 (18.9%). The CRB1-associated LCA showed a pigmented fundus; the RDH12-associated LCA featured macular atrophy. Our results revealed that CRB1 and RPGRIP1 genes occupied a greater proportion in the western Chinese population. The proportion of these two genes was similar in other regions of China as well. The difference existed in a larger proportion of RDH12-associated LCA in the western Chinese population.Conclusions: The new findings in our study group polished the spectrum of the novel mutations and phenotypes of LCA with regional and ethnic variations. This comprehensive database can provide essential information for gene therapies.

Objective The current methods used to assess visual function in blind retinitis pigmentosa (RP) patients are mostly subjective. We aimed to identify effective, objective methods. Methods We enrolled patients diagnosed with blindness associated with RP; we finally selected 26 patients (51 eyes) with a visual field radius less than 10 degrees and divided them into the following 4 groups by best-corrected visual acuity (BCVA): group 1, no light perception (NLP, 4 eyes); group 2, light perception (LP, 12 eyes); group 3, hand movement or finger counting (faint form perception, FFP, 22 eyes); and group 4, BCVA from 0.1 to 0.8 (form perception, FP, 13 eyes). All patients underwent optometry, optical coherence tomography (OCT), color fundus photography, fundus autofluorescence (FAF), full field electroretinography (ffERG), pattern electroretinography (PERG), multifocal electroretinography (mf-ERG), pattern visual evoked potential (PVEP), flash visual evoked potential (FVEP), and pupillary light response (PLR) assessments. Five patients in groups 1, 2, and 3 (1, 2, and 2 subjects, respectively) underwent functional magnetic resonance imaging (fMRI) scans and were compared with five healthy subjects. Results The outer plexiform layer was thinner in group 1, and the outer nuclear layer was thinner in groups 1 and 2. The ffERG, PERG, and mf-ERG findings were unrecordable in all four groups. The P2 amplitude of the FVEP was significantly lower in groups 1 and 2, while the P100 amplitude of the PVEP was higher in groups 2, 3 and 4 than in group 1. After white- and blue-light stimuli, the PLR thresholds in the patients without form perception were significantly higher. The threshold of the PLR stimulated by blue and white light was negatively correlated with the amplitudes of P2 and P100. Moreover, the fMRI findings showed that some RP patients have significant visual cortex activation in response to certain types of stimulation. However, statistical analysis was not performed because of the small number of cases. Conclusions OCT, VEP, PLR and fMRI assessments can evaluate residual visual pathway function in blind RP patients. Significance Our study may have clinical significance for the potential prediction of RP patient prognoses and the effects after clinical trials.

Aim To assess the biosafety of a poly(acrylamide-co-sodium acrylate) hydrogel (PAH) as a 3D-printed intraocular lens (IOL) material. Methods The biosafety of PAH was first evaluated in vitro using human lens epithelial cells (LECs) and the ARPE19 cell line, and a cell counting kit-8 (CCK-8) assay was performed to investigate alterations in cell proliferation. A thin film of PAH and a conventional IOL were intraocularly implanted into the eyes of New Zealand white rabbits respectively, and a sham surgery served as control group. The anterior segment photographs, intraocular pressure (IOP), blood parameters and electroretinograms (ERG) were recorded. Inflammatory cytokines in the aqueous humor, such as TNFα and IL-8, were examined by ELISA. Cell apoptosis of the retina was investigated by TUNEL assay, and macroPAHge activation was detected by immunostaining. Results PAH did not slow cell proliferation when cocultured with human LECs or ARPE19 cells. The implantation of a thin film of a 3D-printed IOL composed of PAH did not affect the IOP, blood parameters, ERG or optical structure in any of the three experimental groups (n=3 for each). Both TNFα and IL-8 in the aqueous humor of PAH group were transiently elevated 1wk post-operation and recovered to normal levels at 1 and 3mo post-operation. Iba1+ macroPAHges in the anterior chamber angle in PAH group were increased markedly compared to those of the control group; however, there was no significant difference compared to those in the IOL group. Conclusion PAH is a safe material for 3D printing of personal IOLs that hold great potential for future clinical applications.

Retinitis pigmentosa (RP) is a hereditary retinal degeneration disease with no effective therapeutic approaches. Inflammatory and immune disorders are thought to play an important role in the pathogenesis of RP. Human umbilical cord mesenchymal stem cells (UCMSCs), with multiple biological functions such as anti-inflammation and immunoregulation, have been applied in different systemic diseases. We conducted a phase I/II clinical trial aiming to evaluate the safety and efficacy of intravenous administration of UCMSCs in advanced RP patients. All 32 subjects were intravenously infused with one dose of 10

Retinitis pigmentosa initially presents as night blindness owing to defects in rods, and the secondary degeneration of cones ultimately leads to blindness. Previous studies have identified active roles of microglia in the pathogenesis of photoreceptor degeneration in RP. However, the contribution of microglia to photoreceptor degeneration remains controversial, partly due to limited knowledge of microglial phenotypes during RP.

Objectives To evaluate the long‐term biosafety and efficacy of transplantation of human embryonic stem cells‐derived retinal pigment epithelial (hESC‐RPE) cells in early‐stage of Stargardt macular degeneration (STGD1). Materials and methods Seven patients participated in this prospective clinical study, where they underwent a single subretinal transplantation of 1 × 105 hESC‐RPE cells in one eye, whereas the fellow eye served as control. These patients were reassessed for a 60‐month follow‐up through systemic and ophthalmic examinations. Results None of the patients experienced adverse reactions systemically or locally, except for two who had transiently high intraocular pressure post‐operation. Functional assessments demonstrated that all of the seven operated eyes had transiently increased or stable visual function 1‐4 months after transplantation. At the last follow‐up visit, two of the seven eyes showed visual function loss than the baseline; however, one of them showed a stable visual acuity when compared with the change of fellow eye. Obvious small high reflective foci in the RPE layer were displayed after the transplantation, and maintained until the last visit. Interestingly, three categories of patients who were classified based on autofluorescence, exhibited distinctive patterns of morphological and functional change. Conclusions Subretinal transplantation of hESC‐RPE in early‐stage STGD1 is safe and tolerated in the long term. Further investigation is needed for choosing proper subjects according to the multi‐model image and function assessments., Transplantation of human embryonic stem cells‐derived retinal pigment epithelial (hESC‐RPE) cells in the patients with early stage of STGD1 is safe and tolerated in the long term. Objective visual functions test after transplantation were mostly remained as the preoperative levels or improved transiently in some cases. Morphology test showed pigmentation in the fundus and high reflective foci in the RPE layer by OCT. Moreover, a new fundus autofluorescence classification was applied to group the subjects for stem cell transplantation and monitored the clinical outcomes.

Background Human umbilical cord mesenchymal stem cells (UCMSCs) transplantation is a promising therapy for the treatment of retinitis pigmentosa (RP). However, intravenously infused cells may be blocked in the lung, increasing the risk of vascular obstruction, which needs to be optimized to further improve safety and efficacy. Methods We derived small UCMSCs (S-UCMSCs) from filtering UCMSCs with a 10-μm filter, and compared with UCMSCs by flow cytometry, directional differentiation culture and transcriptome sequencing. Then the S-UCMSCs and UCMSCs were intravenously infused in the Royal College Surgeons (RCS) rats to evaluate the safety and the efficacy. Results The diameter of S-UCMSCs ranged from 5.568 to 17.231 μm, with an average diameter of 8.636 ± 2.256 μm, which was significantly smaller than that of UCMSCs. Flow cytometry, immunofluorescence and transcriptome sequencing demonstrated that the S-UCMSCs and UCMSCs were the same kind of MSCs, and the S-UCMSCs were more proliferative. After the S-UCMSCs and UCMSCs were intravenously infused into the Royal College of Surgeons (RCS) rats at a dose of 1 × 106 cells/rat, the S-UCMSCs blocked in the lungs were significantly fewer and disappeared more quickly than UCMSCs. The b wave of the flash electroretinogram was improved at 7 d, and the retinal outer nuclear layer thickness was thicker at 7 d and 14 d. The expression level of inflammation was inhibited, and the expression level of neurotrophic factors was upregulated in the retina and serum after transplantation. Conclusions S-UCMSCs intravenous infusion was safer than UCMSCs and could delay retinal degeneration and protect visual function in RCS rats, which may be a preferable therapeutic approach for RP.

To study whether ectopic human melanopsin (hMel) in retinal ganglion cells (RGCs) could restore the visual function in end-stage retinal degeneration, AAV2/8-CMV-hMel/FYP was injected into the intravitreal space of Royal College of Surgeons (RCS) rats. It was observed that ectopic hMel/yellow fluorescent protein (YFP) was dominantly expressed in the RGCs of the RCS rat retinae. At 30-45 days after administration of AAV2/8-CMV-hMel/FYP in RCS rats, the flash visual evoked potentials and behavioral results demonstrated that visual function was significantly improved compared to that in the control group, while no improvement in flash electroretinography was observed at this time point. To translate this potential therapeutic approach to the clinic, the safety of viral vectors in the retinae of normal macaques was then studied, and the expression profile of exogenous hMel with/without internal limiting membrane peeling was compared before viral vector administration. The data revealed that there was no significant difference in the number of RGCs containing exogenous hMel/YFP between the two groups. Whole-cell patch-clamp recordings demonstrated that the hMel/YFP-positive RGCs of the macaque retinae reacted to the intense light stimulation, generating inward currents and action potentials. This result confirms that the ectopic hMel expressed in RGCs is functional. Moreover, the introduction of AAV2/8-CMV-hMel/FYP does not cause detectable pathological effects. Thus, this study suggests that AAV2/8-CMV-hMel/FYP administration without internal limiting membrane peeling is safe and feasible for efficient transduction and provides therapeutic benefits to restore the visual function of patients suffering photoreceptor loss.

We report the results of molecular screening in 121 patients with suspected hereditary optic neuropathies. The 34 primary and 9 secondary LHON mutations were screened in all the patients. In the familial cases, OPA1 was also tested when negative finding for the mtDNA mutations screening. Molecular defects were identified in 35 patients (28.9% of screened patients). Among these, 33 patients (94.3%) had an mtDNA mutation, including m.11778G > A (69.7%), m.14484 T > C, m.3460G > A, m.3635G > A, m.14502 T > C and three secondary mutations m.3316G > A, m.3394 T > C, m.3497C > T. Two novel OPA1 mutations, c.1301 T > G (p.Leu434Arg) and c.985-1G > A (IVS9-1G > A), were also detected in families with the evidence of father-to-son transmission. In conclusion, we reported the results of the molecular screening of 121 patients with hereditary optic neuropathies from southwest of China. Our results highlight the importance of investigating LHON-causing mtDNA mutations and OPA1 mutations in cases of suspected hereditary optic neuropathy.

Hereditary eye diseases have gradually become the main cause of refractory or incurable binocular blindness, a large number of which have no effective treatment. In order to further popularize and promote the ability of diagnosis, treatment, and prevention of hereditary eye diseases in China, the Chinese Eye Genetics Consortium (CEGC) is established, which is attached to the Global Eye Genetics Consortium (GEGC). On June 28, 2018, “The Founding Conference the Chinese Arm of GEGC” was grandly held in Chongqing, China. CEGC includes the Chinese eye genetic disease research collective, the Chinese eye genetic disease experts collective, and the Chinese eye genetic disease prevention and treatment collective. The consortium aims to carry out research on the prevention and treatment of eye genetic diseases, formulate guidelines and consensus for clinical diagnosis of eye genetic diseases, support and cultivate professional talents, and promote international exchanges and cooperation.

Retinal degenerative diseases (RDDs) are the leading causes of blindness and currently lack effective treatment. Cytotherapy has become a promising strategy for RDDs. The transplantation of olfactory ensheathing cells (OECs) or neural stem cells (NSCs) has recently been applied for the experimental treatment of RDDs. However, the long-term outcomes of single-cell transplantation are poor. The combined transplantation of multiple types of cells might achieve better effects. In the present study, OECs [containing olfactory nerve fibroblasts (ONFs)] and NSCs were cotransplanted into the subretinal space of Royal College of Surgeons (RCS) rats. Using electroretinogram (ERG), immunofluorescence, Western blot, and in vitro Transwell system, the differences in the electrophysiological and morphological changes of single and combined transplantation as well as the underlying mechanisms were explored at 4, 8, and 12 weeks postoperation. In addition, using the Transwell system, the influence of OECs on the stemness of NSCs was discovered. Results showed that, compared to the single transplantation of OECs or NSCs, the combined transplantation of OECs and NSCs produced greater improvements in b-wave amplitudes in ERGs and the thickness of the outer nuclear layer at all three time points. More endogenous stem cells were found within the retina after combined transplantation. Glial fibrillary acidic protein (GFAP) expression decreased significantly when NSCs were cotransplanted with OECs. Both the vertical and horizontal migration of grafted cells were enhanced in the combined transplantation group. Meanwhile, the stemness of NSCs was also better maintained after coculture with OECs. Taken together, the results suggested that the combined transplantation of NSCs and OECs enhanced the improvement in retinal protection in RCS rats, providing a new strategy to treat RDDs in the future.

Objective. To review retrospectively the electrophysiological and structural changes in 13 Chinese patients with Leber hereditary optic neuropathy (LHON). Methods. 26 eyes of 13 patients with a genetically confirmed diagnosis of LHON were categorized into two groups according to the duration of the disease: group 1 (duration less than 3 months) and group 2 (duration between 3 months and 18 years). Clinical history, comprehensive visual electrophysiology, optical coherence tomography (OCT), and color fundus photography were performed. Results. Fundoscopy showed optic disc hyperemia in group 1 and optic atrophy in group 2. OCT measures of retinal nerve fiber layer (RNFL) thickness around the optic disc and surrounding macula were normal in group 1 but reduced in group 2 (10 of 10 eyes). The thickness of the retinal ganglion cell layer (GCL) plus inner plexiform layer (IPL) surrounding the macula reduced significantly in group 1 and group 2 compared with a healthy control group. Pattern ERG (PERG) P50 amplitude was normal, but the N95/P50 ratio reduced in most of group 1 (4 of 5 eyes) and in all of group 2 (11 eyes). PERG P50 peak time was abnormally short in group 2. Multifocal electroretinography (mfERG) showed subnormal responses associated with ring 1 (the central area) and ring 2 in group 1 and reductions in rings 1, 2, and 3 in group 2. Conclusion. The study highlights differences in retinal structure and function between the acute and chronic stages of LHON in a group of Chinese patients. There is PERG evidence of retinal ganglion cell dysfunction and OCT evidence of GCL + IPL thinning in both groups, but there is additional peripapillary RNFL loss in the chronic stage, associated with more severe RGC dysfunction. There is multifocal ERG evidence of localized macular dysfunction in both acute and chronic groups. The study highlights the importance of comprehensive electrophysiological and structural assessments of the retina in LHON and is pertinent to studies that aim to monitor disease progression or the effects of future therapeutic interventions.

Background: The USH2A gene encodes usherin, a basement membrane protein that is involved in the development and homeostasis of the inner ear and retina. Mutations in USH2A are linked to Usher syndrome type II (USH II) and non-syndromic retinitis pigmentosa (RP). Molecular diagnosis can provide insight into the pathogenesis of these diseases, facilitate clinical diagnosis, and identify individuals who can most benefit from gene or cell replacement therapy. Here, we report 21 pathogenic mutations in the USH2A gene identified in 11 Chinese families by using the targeted next-generation sequencing (NGS) technology. Methods: In all, 11 unrelated Chinese families were enrolled, and NGS was performed to identify mutations in the USH2A gene. Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families. Results: We identified 21 pathogenic mutations, of which 13, including 5 associated with non-syndromic RP and 8 with USH II, have not been previously reported. The novel variants segregated with disease phenotype in the affected families and were absent from the control subjects. In general, visual impairment and retinopathy were consistent between the USH II and non-syndromic RP patients with USH2A mutations. Conclusions: These findings provide a basis for investigating genotype–phenotype relationships in Chinese USH II and RP patients and for clarifying the pathophysiology and molecular mechanisms of the diseases associated with USH2A mutations.

Graphene and its derivatives are widely used for a variety of industrial, biomedical, and environmental applications. However, the potential harm caused by exposure of the eyes to graphene-based nanomaterials is scarce. Given the potential for these materials to be used in multiple applications, there is a pressing need to evaluate their ocular toxicity, and understand the relationships between their physico-chemical properties and the resulting toxicity. In this study, the toxicity of PEGylated graphene oxide (PEG-GO) with differing oxidation levels and/or surface charges (positive, negative and neutral charge) was evaluated using two in-vitro models of the eye: primary human corneal epithelial cells and human retinal capillary endothelial cells. The results showed that oxidation level, but not surface charge, had a pivotal effect on the toxicity of graphene-based nanomaterials. Typically, PEG-GO sample with a higher oxidation level caused more serious cytotoxicity than those with a lower oxidation level. Furthermore, by analysis of global gene expression profiles, we found that the foremost cellular response to PEG-GO sample with a high oxidation level was the oxidative stress response. Next, via exploring the underlying molecular mechanism of oxidative stress-induced cytotoxicity, we showed that PEG-GO sample with a high degree of oxidation induced reactive oxygen species (ROS) via NDUFB9-mediated biological pathway. This work has significant implications for design of safe graphene-based nanomaterials for biomedical applications.

Diabetic retinopathy (DR), one of the leading causes of vision loss worldwide, is characterized by neurovascular disorders. Emerging evidence has demonstrated retinal neurodegeneration in the early pathogenesis of DR, and no treatment has been developed to prevent the early neurodegenerative changes that precede detectable microvascular disorders. Bone marrow CD133+ stem cells with revascularization properties exhibit neuroregenerative potential. However, whether CD133+ cells can ameliorate the neurodegeneration at the early stage of DR remains unclear. In this study, mouse bone marrow CD133+ stem cells were immunomagnetically isolated and analyzed for the phenotypic characteristics, capacity for neural differentiation, and gene expression of neurotrophic factors. After being labeled with enhanced green fluorescent protein, CD133+ cells were intravitreally transplanted into streptozotocin (STZ)-induced diabetic mice to assess the outcomes of visual function and retina structure and the mechanism underlying the therapeutic effect. We found that CD133+ cells co-expressed typical hematopoietic/endothelial stem/progenitor phenotypes, could differentiate to neural lineage cells, and expressed genes of robust neurotrophic factors in vitro. Functional analysis demonstrated that the transplantation of CD133+ cells prevented visual dysfunction for 56 days. Histological analysis confirmed such a functional improvement and showed that transplanted CD133+ cells survived, migrated into the inner retina (IR) over time and preserved IR degeneration, including retina ganglion cells (RGCs) and rod-on bipolar cells. In addition, a subset of transplanted CD133+ cells in the ganglion cell layer differentiated to express RGC markers in STZ-induced diabetic retina. Moreover, transplanted CD133+ cells expressed brain-derived neurotrophic factors (BDNFs) in vivo and increased the BDNF level in STZ-induced diabetic retina to support the survival of retinal cells. Based on these findings, we suggest that transplantation of bone marrow CD133+ stem cells represents a novel approach to ameliorate visual dysfunction and the underlying IR neurodegeneration at the early stage of DR.

Olfactory ensheathing cells (OECs) have been shown to be a leading candidate in cell therapies for central nervous system (CNS) injuries and neurodegenerative diseases. Rapid clearance of neuron debris can promote neuronal survival and axonal regeneration in CNS injuries and neurodegenerative diseases. The phagocytic removal of neuron debris by OECs has been shown to contribute to neuronal outgrowth. However, the precise molecular and cellular mechanisms of phagocytic removal of neuron debris by OECs have not been explored. In this study, we found that OECs secreted anti-inflammatory cytokine transforming growth factor β1 (TGF-β1) during the phagocytic removal of neuron debris. TGF-β1 enhanced phagocytic activity of OECs through regulating integrin/MFG-E8 signaling pathway. In addition, TGF-β1 shifted OECs towards a flattened shape with increased cellular area, which might also be involved in the enhancement of phagocytic activity of OECs. Furthermore, the removal of neuron debris by OECs affected neuronal survival and outgrowth. TGF-β1 enhanced the clearance of neuron debris by OECs and increased neuronal survival. These results reveal the role and mechanism of TGF-β1 in enhancing the phagocytic activity of OECs, which will update the understanding of phagocytosis of OECs and improve the therapeutic use of OECs in CNS injuries and neurodegenerative diseases.

// Langyue Xue 1, 2 , Yuxiao Zeng 1, 2 , Qiyou Li 1, 2 , Yijian Li 1, 2 , Zhengya Li 1, 2 , Haiwei Xu 1, 2 and Zhengqin Yin 1, 2 1 Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038, China 2 Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038, China Correspondence to: Haiwei Xu, email: haiweixu2001@163.com Zhengqin Yin, email: qinzyin@aliyun.com Keywords: light damage, photoreceptor, olfactory ensheathing cells, reactive oxygen species Received: March 28, 2017 Accepted: August 08, 2017 Published: October 16, 2017 ABSTRACT There is still not an effective treatment for continuous retinal light exposure and subsequent photoreceptor degeneration. Olfactory ensheathing cell (OEC) transplantation has been shown to be neuroprotective in spinal cord, and optic nerve injury and retinitis pigmentosa. However, whether OECs protect rat photoreceptors against light-induced damage and how this may work is unclear. Thus, to elucidate this mechanism, purified rat OECs were grafted into the subretinal space of a Long-Evans rat model with light-induced photoreceptor damage. Light exposure decreased a- and b- wave amplitudes and outer nuclear layer (ONL) thickness, whereas the ONL of rats exposed to light for 24 h after having received OEC transplants in their subretinal space was thicker than the PBS control and untreated groups. A- and b- wave amplitudes from electroretinogram of OEC-transplanted rats were maintained until 8 weeks post OEC transplantation. Also, transplanted OECs inhibited formation of reactive oxygen species in retinas exposed to light. In vitro experiments showed that OECs had more total antioxidant capacity in a co-cultured 661W photoreceptor cell line, and cells were protected from damage induced by hydrogen-peroxide. Thus, transplanted OECs preserved retinal structure and function in a rat model of light-induced degeneration by suppressing retinal oxidative stress reactions.

Retinal oscillatory potentials (OPs) consist of a series of relatively high-frequency rhythmic wavelets, superimposed onto the ascending phase of the b-wave of the electroretinogram (ERG). However, the origin of OPs is uncertain and methods of measurement of OPs are diverse. In this study, we first isolated OPs from the rat ERG and fitted them with Gabor functions and found that the envelope of the OP contained information about maximum amplitude and time-to-peak to enable satisfactory quantification of the later OPs. And the OP/b-wave ratio should be evaluated to exclude an effect of the b-wave on the OPs. Next, we recorded OPs after intravitreal injection of 2-amino-4-phosphonobutyric acid (APB), tetrodotoxin (TTX), γ-aminobutyric acid (GABA), strychnine (STR), SR95531 (SR), isoguvacine (ISO), (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) and GABA+TPMPA. We showed that GABA and APB only removed the later OPs, when compared to control eyes. TTX delayed the peak time, and STR, SR and ISO reduced the amplitude of OPs. TPMPA delayed the peak time but increased the ratio of OPs to b-wave. Furthermore, administration of combined GABA and TPMPA caused the later OPs to increase in amplitude with time, compared with those after delivery of GABA alone. Finally, we observed that GABAc and glycine receptors contributed to a low-frequency component of the OPs, while GABAa contributed to both components. These results suggest that the early components of the OPs are mainly generated by the photoreceptors, whilst the later components are mainly regulated by GABAa, GABAc and glycine receptors.

The inflammatory microenvironment in the retina plays a vital role in the pathogenesis and progression of retinitis pigmentosa (RP). Microglial inflammatory cytokines production leads to gliosis and apoptosis of retinal neurons, and ultimately, visual loss. Cell-based therapies using grafted olfactory ensheathing cells (OECs) have demonstrated modulation of degenerative microenvironments in the central nervous system (CNS), in a number of animal models. However, mechanisms by which grafted OECs can reduce degeneration in the retina are not well understood. In the present study, we set up an in vitro OEC/BV2 microglia co-culture system, and an in vivo royal college of surgeons (RCS) rat model, used cell transplantation, immunohistochemistry, RT-PCR, western blot to explore the mechanisms by which OECs affect expression of pro- or anti-inflammatory cytokines and polarization of M(IL-6) and M(Arg1) type microglial activation in the retina. We found that compared with the LPS (Lipopolysaccharide) and olfactory nerve fibroblast (ONF), the OEC and BV2 co-culture group modulate microglial cytokines releasing toward the anti-inflammation, and away from the pro-inflammation, which was followed by higher IL-4 and IL-10 and lower TNF-a and IL-6 in their expression levels. In vivo, the transplantation group significantly reduced activated resident microglia/infiltrated macrophage, and expression of pro-inflammatory cytokines in RCS rats retina, increased anti-inflammatory cytokines in transplantation area. Additionally, we found that OECs expressed SOCS3 and down-regulated the JAK2/STAT3 (Janus Kinase 2/Signal Transducer and Activator of Transcription 3) pathway. Thirdly, OEC transplantation reduced Caspase-3 expression, protected inner retinal neurons and photoreceptors and therefore, delayed the visual function degeneration. In conclusion, our data suggest that OECs delay retinal degeneration in RP, at least in part through immunomodulation of microglia via the JAK/STAT pathway.

Background Retinal degeneration (RD) is characterized by progressive photoreceptor degeneration, and emerging evidence has demonstrated that activated microglia-mediated inflammation exacerbates the progression of RD. Lipoxin A4 (LXA4) is an endogenous neuroprotective lipid mediator, but the potential therapeutic roles of LXA4 in RD have not been evaluated. Methods Electroretinogram (ERG) recordings and behavioral tests were used to analyze whether the intravitreal injection (IVI) of LXA4 restored visual function in RD1 mice. Immunostaining, qPCR, western blotting and mouse cytokine arrays using an ex-vivo retinal explant model were successively performed to explore the mechanisms underlying the effects of LXA4. Results The key rate-limiting enzyme in LXA4 biosynthesis and the LXA4 receptor were substantially downregulated in end-stage RD1 retinas. LXA4 maintained visual function in RD1 mice from postnatal days 15–21 (PN15 to PN21). Moreover, LXA4 modulated microglial activities, significantly inhibited proinflammatory gene expression, and thereby attenuated photoreceptor apoptosis. Conclusions LXA4 delayed the progression of RD, and thus, the use of LXA4 might be a novel approach for ameliorating dysfunction in neurodegenerative disorders.

Stem cell therapy may replace lost photoreceptors and preserve residual photoreceptors during retinal degeneration (RD). Unfortunately, the degenerative microenvironment compromises the fate of grafted cells, demanding supplementary strategies for microenvironment regulation. Donor cells with both proper regeneration capability and intrinsic ability to improve microenvironment are highly desired. Here, we use cell surface markers (C-Kit+/SSEA4−) to effectively eliminate tumorigenic embryonic cells and enrich retinal progenitor cells (RPCs) from human embryonic stem cell (hESC)-derived retinal organoids, which, following subretinal transplantation into RD models of rats and mice, significantly improve vision and preserve the retinal structure. We characterize the pattern of integration and materials transfer following transplantation, which likely contribute to the rescued photoreceptors. Moreover, C-Kit+/SSEA4− cells suppress microglial activation, gliosis and the production of inflammatory mediators, thereby providing a healthier host microenvironment for the grafted cells and delaying RD. Therefore, C-Kit+/SSEA4− cells from hESC-derived retinal organoids are a promising therapeutic cell source., Stem cell transplantation to treat retinal degeneration could be limited by the degenerative microenvironment. Here, the authors show that C-Kit+/SSEA4– progenitor cells enriched from human embryonic stem cell derived retinal organoids protect retinal structure, suppress microglial activation, gliosis and inflammation.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

Graphene and its derivatives are the new carbon nanomaterials with the prospect for great applications in electronics, energy storage, biosensors and medicine. However, little is known about the toxicity of graphene or its derivatives in the case of occasional or repeated ocular exposure. We performed in vitro and in vivo studies to evaluate the toxicity of graphene oxide (GO) exposure to the eye. Primary human corneal epithelium cells (hCorECs) and human conjunctiva epithelium cells (hConECs) were exposed to GO (12.5-100 μg/mL). Acute GO exposure (2 h) did not induce cytotoxicity to hCorECs. However, short-term GO exposure (24 h) exerted significant cytotoxicity to hCorECs and hConECs with increased intracellular reactive oxygen species (ROS). Glutathione (GSH) reduced the GO-induced cytotoxicity. We further performed acute eye irritation tests in albino rabbits according to the Organization for Economic Cooperation and Development (OECD) guidelines, and the rabbits did not exhibit corneal opacity, conjunctival redness, abnormality of the iris, or chemosis at any time point after the instillation of 100 μg/mL of GO. However, 5-day repeated GO exposure (50 and 100 μg/mL) caused reversible mild corneal opacity, conjunctival redness and corneal epithelium damage to Sprague-Dawley rats, which was also alleviated by GSH. Therefore, our study suggests that GO-induced time- and dose-dependent cytotoxicity to hCorECs and hConECs via oxidative stress. Occasional GO exposure did not cause acute eye irritation; short-term repeated GO exposure generally resulted in reversible damage to the eye via oxidative stress, which may be alleviated by the antioxidant GSH.

Retinal regeneration and repair are severely impeded in higher mammalian animals. Although Muller cells can be activated and show some characteristics of progenitor cells when injured or under pathological conditions, they quickly form gliosis scars. Unfortunately, the basic mechanisms that impede retinal regeneration remain unknown. We studied retinas from Royal College of Surgeon (RCS) rats and found that let-7 family molecules, let-7e and let-7i, were significantly overexpressed in Muller cells of degenerative retinas. It demonstrated that down-regulation of the RNA binding protein Lin28B was one of the key factors leading to the overexpression of let-7e and let-7i. Lin28B ectopic expression in the Muller cells suppressed overexpression of let-7e and let-7i, stimulated and mobilized Muller glia de-differentiation, proliferation, promoted neuronal commitment, and inhibited glial fate acquisition of de-differentiated Muller cells. ERG recordings revealed that the amplitudes of a-wave and b-wave were improved significantly after Lin28B was delivered into the subretinal space of RCS rats. In summary, down-regulation of Lin28B as well as up-regulation of let-7e and let-7i may be the main factors that impede Muller cell de-differentiation and proliferation in the retina of RCS rats.

AIM: To explore whether ectopic expression of human melanopsin can effectively and safely restore visual function in rd1 mice. METHODS: Hematoxylin-eosin staining of retinal sections from rd1 mice was used to detect the thickness of the outer nuclear layer to determine the timing of surgery. We constructed a human melanopsin-AAV2/8 viral vector and injected it into the subretinal space of rd1 mice. The Phoenix Micron IV system was used to exclude the aborted injections, and immunohistochemistry was used to validate the ectopic expression of human melanopsin. Furthermore, visual electrophysiology and behavioral tests were used to detect visual function 30 and 45d after the injection. The structure of the retina was compared between the human melanopsin-injected group and phosphate buffer saline (PBS)-injected group. RESULTS: Retinas of rd1 mice lost almost all of their photoreceptors on postnatal day 28 (P28). We therefore injected the human melanopsin-adeno-associated virus (AAV) 2/8 viral vector into P30 rd1 mice. After excluding aborted injections, we used immunohistochemistry of the whole mount retina to confirm the ectopic expression of human melanopsin by co-expression of human melanopsin and YFP that was carried by a viral vector. At 30d post-injection, visual electrophysiology and the behavioral test significantly improved. However, restoration of vision disappeared 45d after human melanopsin injection. Notably, human melanopsin-injected mice did not show any structural differences in their retinas compared with PBS-injected mice. CONCLUSION: Ectopic expression of human melanopsin effectively and safely restores visual function in rd1 mice.

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1β, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

Resident microglia are the main immune cells in the retina and play a key role in the pathogenesis of retinitis pigmentosa (RP). Many previous studies on the roles of microglia mainly focused on the neurotoxicity or neuroprotection of photoreceptors, while their contributions to synaptic remodeling of neuronal circuits in the retina of early RP remained unclarified. In the present study, we used Royal College of Surgeons (RCS) rats, a classic RP model characterized by progressive microglia activation and synapse loss, to investigate the constitutive effects of microglia on the synaptic lesions and ectopic neuritogenesis. Rod degeneration resulted in synapse disruption and loss in the outer plexiform layer (OPL) at the early stage of RP. Coincidentally, the resident microglia in the OPL increased phagocytosis and mainly engaged in phagocytic engulfment of postsynaptic mGluR6 of rod bipolar cells (RBCs). Complement pathway might be involved in clearance of postsynaptic elements of RBCs by microglia. We pharmacologically deleted microglia using a CSF1 receptor (CSF1R) inhibitor to confirm this finding, and found that it caused the accumulation of postsynaptic mGluR6 levels and increased the number and length of ectopic dendrites in the RBCs. Interestingly, the numbers of presynaptic sites expressing CtBP2 and colocalized puncta in the OPL of RCS rats were not affected by microglia elimination. However, sustained microglial depletion led to progressive functional deterioration in the retinal responses to light in RCS rats. Based on our results, microglia mediated the remodeling of RBCs by phagocytosing postsynaptic materials and inhibiting ectopic neuritogenesis, contributing to delay the deterioration of vision at the early stage of RP.

Purpose To identify any novel mutations in CYP4V2 in 85 Chinese families with Bietti corneoretinal crystalline dystrophy (BCD) by using next-generation sequencing, and to summarize the mutation spectrum in this population, along with any genotype-phenotype correlations. Methods A total of 90 patients with BCD from 85 unrelated Chinese families were recruited. All probands were analyzed by using gene chip-based next-generation sequencing, to capture and sequence all the exons of 57 known hereditary retinal degeneration-associated genes. The candidate variants were validated with PCR and Sanger sequencing. Results Twenty-eight mutations were detected in all patients, including thirteen novel mutations (five missense, six deletions, one splicing and one frame-shift mutations) and 15 previously reported mutations. Mutations in 64 patients were inherited from their parents, while three patients had de novo mutations. c.802–8_810del17insGC was the most common mutation, accounting for 78% of the mutations. Although 16 patients were homozygous at this site, the clinical features of all 16 patients were highly heterogeneous. Conclusions These results expand the spectrum of mutations in CYP4V2, and suggest that mutations in CYP4V2 may be common in the Chinese population. The phenotype of patients with the homozygous mutation (hom.c.802–8_810del17insGC) is highly heterogeneous.

AIM: To evaluate the intrinsic excitability of retinal ganglion cells (RGCs) in degenerated retinas. METHODS: The intrinsic excitability of various morphologically defined RGC types using a combination of patch-clamp recording and the Lucifer yellow tracer in retinal whole-mount preparations harvested from Royal College of Surgeons (RCS) rats, a common retinitis pigmentosa (RP) model, in a relatively late stage of retinal degeneration (P90) were investigated. Several parameters of RGC morphologies and action potentials (APs) were measured and compared to those of non-dystrophic control rats, including dendritic stratification, dendritic field diameter, peak amplitude, half width, resting membrane potential, AP threshold, depolarization to threshold, and firing rates. RESULTS: Compared with non-dystrophic control RGCs, more depolarizations were required to reach the AP threshold in RCS RGCs with low spontaneous spike rates and in RCS OFF cells (especially A2o cells), and RCS RGCs maintained their dendritic morphologies, resting membrane potentials and capabilities to generate APs. CONCLUSION: RGCs are relatively well preserved morphologically and functionally, and some cells are more susceptible to decreased excitability during retinal degeneration. These findings provide valuable considerations for optimizing RP therapeutic strategies.

Diabetic retinopathy (DR), one of the leading causes of vision loss worldwide, is characterized by neurovascular disorders. Emerging evidence has demonstrated retinal neurodegeneration in the early pathogenesis of DR, and no treatment has been developed to prevent the early neurodegenerative changes that precede detectable microvascular disorders. Bone marrow CD133+ stem cells with revascularization properties exhibit neuroregenerative potential. However, whether CD133+ cells can ameliorate the neurodegeneration at the early stage of DR remains unclear. In this study, mouse bone marrow CD133+ stem cells were immunomagnetically isolated and analyzed for the phenotypic characteristics, capacity for neural differentiation, and gene expression of neurotrophic factors. After being labeled with enhanced green fluorescent protein, CD133+ cells were intravitreally transplanted into streptozotocin (STZ)-induced diabetic mice to assess the outcomes of visual function and retina structure and the mechanism underlying the therapeutic effect. We found that CD133+ cells co-expressed typical hematopoietic/endothelial stem/progenitor phenotypes, could differentiate to neural lineage cells, and expressed genes of robust neurotrophic factors in vitro. Functional analysis demonstrated that the transplantation of CD133+ cells prevented visual dysfunction for 56 days. Histological analysis confirmed such a functional improvement and showed that transplanted CD133+ cells survived, migrated into the inner retina (IR) over time and preserved IR degeneration, including retina ganglion cells (RGCs) and rod-on bipolar cells. In addition, a subset of transplanted CD133+ cells in the ganglion cell layer differentiated to express RGC markers in STZ-induced diabetic retina. Moreover, transplanted CD133+ cells expressed brain-derived neurotrophic factors (BDNFs) in vivo and increased the BDNF level in STZ-induced diabetic retina to support the survival of retinal cells. Based on these findings, we suggest that transplantation of bone marrow CD133+ stem cells represents a novel approach to ameliorate visual dysfunction and the underlying IR neurodegeneration at the early stage of DR.

Stem cell therapy may provide a safe and promising treatment for retinal diseases. Wet age-related macular degeneration (wet-AMD) is a leading cause of blindness in China. We developed a clinical-grade human embryonic stem cell (hESC) line, Q-CTS-hESC-2, under xeno-free conditions that differentiated into retinal pigment epithelial cells (Q-CTS-hESC-2-RPE). A clinical trial with three wet-AMD patients was initiated in order to study the safety and tolerance to Q-CTS-hESC-2-RPE cell transplants. The choroidal neovascularization membrane was removed and then a suspension of 1 × 106 Q-CTS-hESC-2-RPE cells were injected into a subfoveal pocket. The patients were followed for 12 months during which no adverse effects resulting from the transplant were observed. Anatomical evidence suggested the existence of new RPE-like cell layer in the previously damaged area. Visual and physiological testing indicated limited functional improvement, albeit to different degrees between patients. This study provides some promising early results concerning the use of transplanted hESC-RPE cells to alleviate wet-AMD.

Background. To evaluate the treatment outcomes of patients with punctate inner choroidopathy (PIC) and secondary choroidal neovascularization (CNV). Methods. This is a retrospective study of 24 eyes in 22 patients suffering from PIC with CNV. Patients were treated with intravitreal ranibizumab monotherapy (14 eyes) or combined oral corticosteroid and intravitreal ranibizumab therapy (corticosteroid-ranibizumab group, 10 eyes). Mean follow-up duration was 24.0 months. We evaluated best-corrected visual acuity (BCVA), fundus autofluorescence, fluorescein angiography, indocyanine green angiography, and optical coherence tomography, before and after treatment. The following variables were compared between groups: number of intravitreal ranibizumab injections, BCVA, recurrence of CNV, and change in PIC lesions. Results. The ranibizumab monotherapy group received an average of 3 intravitreal ranibizumab injections; mean logMAR visual acuity improvement was 0.34, and 8 eyes developed recurrent CNV during follow-up. The corticosteroid-ranibizumab group received an average of 1.9 intravitreal ranibizumab injections; mean logMAR visual acuity improvement was 0.61, and there was no recurrence of CNV. Combined corticosteroid-ranibizumab therapy also resulted in better resolution of PIC lesions and fewer new PIC lesions. Conclusion. Both corticosteroid-ranibizumab treatment and ranibizumab monotherapy could significantly improve the vision of PIC patients with CNV. Combined corticosteroid and intravitreal ranibizumab treatment appeared to reduce CNV recurrence and development of new PIC lesions compared with ranibizumab monotherapy.