Small extracellular vesicles of organoid-derived human retinal stem cells remodel Müller cell fate via miRNA: A novel remedy for retinal degeneration.

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.

Transplanted OECs Protect Visual Function by Regulating the Glutamate Metabolic Microenvironment in the Glaucoma Model.

BACKGROUND: Glaucoma is the leading cause of irreversible blindness, and the loss of retinal ganglion cells (RGCs) is the most important pathological feature. During the progression of glaucoma, glutamate content in the optic nerve increases, and glutamate-induced excitotoxicity will aggregate the damage and death of RGCs. We have previously reported that olfactory ensheathing cells (OECs) transplantation preserved the visual function of the glaucoma model but the mechanism is unknown. METHODS: Adult Long-Evans rats were used in the present study and injecting magnetic microspheres was used to establish a glaucoma model in rats. Optokinetic response test and Pattern electroretinogram recording were used to assess the visual functions of rats. RT-PCR, immunofluorescence, and co-culture experiments were performed to investigate the therapeutic effects and mechanisms of OECs for glaucoma. RESULTS: In the glaucoma model, increased glutamate content and the damage of astrocytes (AC) and RGCs were observed. OECs transplantation reduced the glutamate concentration in the optic nerve, alleviated the apoptosis of AC and RGCs, and protected the visual function of the glaucoma model. Furthermore, we found that OECs possessed a stronger capacity to metabolize excessive glutamate compared with AC and Müller glia. OECs could improve the glutamate microenvironment of the optic nerve to prevent AC and RGCs from glutamate-induced excitotoxicity in glaucoma. And the recovery of AC function further supported the survival of RGCs. CONCLUSIONS: We demonstrate that OECs transplantation could play a neuroprotective role by regulating the glutamate microenvironment in glaucoma.

Organoid-derived human retinal progenitor cells promote early dedifferentiation of Müller glia in Royal College of Surgeons rats.

AIM: To explore whether the subretinal transplantation of retinal progenitor cells from human embryonic stem cell-derived retinal organoid (hERO-RPCs) could promote Müller glia dedifferentiation and transdifferentiation, thus improving visual function and delaying retinal degenerative progression. METHODS: hERO-RPCs were subretinally transplanted into Royal College of Surgeons (RCS) rats. Electroretinography (ERG) recording was performed at 4 and 8wk postoperation to assess retinal function. Using immunofluorescence, the changes in outer nuclear layer (ONL) thickness and retinal Müller glia were explored at 2, 4, and 8wk postoperation. To verify the effect of hERO-RPCs on Müller glia in vitro, we cocultured hERO-RPCs with Müller glia with a Transwell system. After coculture, Ki67 staining and quantitative polymerase chain reaction (qPCR) were performed to measure the proliferation and mRNA levels of Müller glia respectively. Cell migration experiment was used to detect the effect of hERO-RPCs on Müller glial migration. Comparisons between two groups were performed by the unpaired Student's t-test, and comparisons among multiple groups were made with one-way ANOVA followed by Tukey's multiple comparison test. RESULTS: The visual function and ONL thickness of RCS rats were significantly improved by transplantation of hERO-RPCs at 4 and 8wk postoperation. In addition to inhibiting gliosis at 4 and 8wk postoperation, hERO-RPCs significantly increased the expression of dedifferentiation-associated transcriptional factor in Müller glia and promoted the migration at 2, 4 and 8wk postoperation, but not the transdifferentiation of these cells in RCS rats. In vitro, using the Transwell system, we found that hERO-RPCs promoted the proliferation and migration of primary rat Müller glia and induced their dedifferentiation at the mRNA level. CONCLUSION: These results show that hERO-RPCs might promote early dedifferentiation of Müller glia, which may provide novel insights into the mechanisms of stem cell therapy and Müller glial reprogramming, contributing to the development of novel therapies for retinal degeneration disorders.

Disease-associated microglial activation prevents photoreceptor degeneration by suppressing the accumulation of cell debris and neutrophils in degenerating rat retinas.

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. Rationale: In this study, we investigated the pathways of microglial activation and its contribution to photoreceptor degeneration in RP. Methods: A classic RP model, Royal College of Surgeons rat, was used to explore the process of microglial activation during the development of RP. An inhibitor of colony-stimulating factor 1 receptor (PLX3397) was fed to RCS rats for sustained ablation of microglia. Immunohistochemistry, flow cytometry, RT-qPCR, electroretinography and RNA-Seq were used to investigate the mechanisms by which activated microglia influenced photoreceptor degeneration. Results: Microglia were gradually activated to disease-associated microglia in the photoreceptor layers of RCS rats. Sustained treatment with PLX3397 ablated most of the disease-associated microglia and aggravated photoreceptor degeneration, including the secondary degeneration of cones, by downregulating the expression of genes associated with photoreceptor function and components and exacerbating the impairment of photoreceptor cell function. Disease-associated microglial activation promoted microglia to engulf apoptotic photoreceptor cell debris and suppressed the increase of infiltrated neutrophils by increasing engulfment and inhibiting CXCL1 secretion by Müller cells, which provided a healthier microenvironment for photoreceptor survival. Conclusions: Our data highlight a key role of disease-associated microglia activation in the suppression of rod and cone degeneration, which reduces secondary damage caused by the accumulation of dead cells and infiltrated neutrophils in the degenerating retina.

Intravenous infusion of small umbilical cord mesenchymal stem cells could enhance safety and delay retinal degeneration in RCS rats.

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.

A phase I clinical trial of human embryonic stem cell-derived retinal pigment epithelial cells for early-stage Stargardt macular degeneration: 5-years' follow-up.

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.

Development-related mitochondrial properties of retinal pigment epithelium cells derived from hEROs.

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.

Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

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.

Molecular genetics with clinical characteristics of Leber congenital amaurosis in the Han population of western China.

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.

Quantitative assessment of visual pathway function in blind retinitis pigmentosa patients.

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.

Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

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.

Biosafety of a 3D-printed intraocular lens made of a poly(acrylamide-co-sodium acrylate) hydrogel in vitro and in vivo.

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.

Fish oil supplementation and repeated macular hemorrhage without choroidal neovascularization: A case report.

Macular hemorrhage can occur spontaneously and repeatedly without choroidal neovascularization or other known lesions associated with myopia. We report a case of repeated myopic macular hemorrhage following fish oil supplementation. A 32-year-old male was referred with newly acquired paracentral scotoma in the left eye. Serial retinal imaging, including fundus photography, fluorescein angiography, and spectral-domain optical coherence tomography were performed. Fundus photography and fluorescein angiography showed a subtle red-colored lesion nasal to the fovea. Optical coherence tomography showed a dome shaped elevation in the ellipsoid zone and interdigitation zone in the left eye. No known ocular risk factors for macular hemorrhage, such as choroidal neovascularization, lacquer cracks, Fuch's spot or choroid thinning or keratoconus were observed. After 2 months without any treatment, the left eye lesion disappeared. However 2 weeks later, another newly developed red-colored lesion close to the left fovea was observed. At that moment, the detailed medical history revealed that the patient had been regularly taking a high dose of commercially available fish oil supplement beginning one month before the first macular hemorrhage. After discontinuation of the fish oil, the second left hemorrhage resolved gradually over the following 8 weeks. No recurrent hemorrhages have been detected at the 12 months follow-up visits. Our observations suggest that the relative value of nutritional supplementation with high doses of fish oil should be cautioned in patients with repetitive retinal hemorrhage.

Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells Maintains and Partially Improves Visual Function in Patients with Advanced Retinitis Pigmentosa.

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 108 UCMSCs and were followed up for 12 months. No serious local or systemic adverse effects occurred in the whole follow-up. Most patients improved their best corrected visual acuity (BCVA) in the first 3 months. The proportions of patients with improved or maintained BCVA were 96.9%, 95.3%, 93.8%, 95.4%, 90.6%, and 90.6% at the 1st, 2nd, 3rd, 6th, 9th, and 12th month follow-up, respectively. Most of the patients (81.3%) maintained or improved their visual acuities for 12 months. The average NEI VFQ-25 questionnaire scores were significantly improved at the third month (P < 0.05). The average visual field sensitivity and flash visual evoked potential showed no significant difference (P = 0.185, P = 0.711). Our results indicated that the intravenous infusion of UCMSCs was safe for advanced RP patients. Most of the patients improved or maintained their visual functions in a long term. The life qualities were improved significantly in the first 3 months, suggesting that the intravenous infusion of UCMSCs may be a promising therapeutic approach for advanced RP patients.

Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia.

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-1ß, 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.

Electrophysiological and Structural Changes in Chinese Patients with LHON.

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.

Combined Transplantation of Olfactory Ensheathing Cells With Rat Neural Stem Cells Enhanced the Therapeutic Effect in the Retina of RCS Rats.

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.

Identification of 13 novel USH2A mutations in Chinese retinitis pigmentosa and Usher syndrome patients by targeted next-generation sequencing.

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.

Novel mutations in CYP4V2 in Bietti corneoretinal crystalline dystrophy: Next-generation sequencing technology and genotype-phenotype correlations.

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.

SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina.

The stem cell factor receptor (SCFR) has been demonstrated to be expressed in the neural retina of mice, rat and human for decades. Previous reports indicated that the SCFR correlates with glia differentiation of late retinal progenitor cells (RPCs), retinal vasculogenesis and homeostasis of the blood-retinal barrier. However, the role of SCF/SCFR signaling in the growth and development of the neural retina (NR), especially in the early embryonic stage, remains poorly understood. Here, we show that SCF/SCFR signaling orchestrates invagination of the human embryonic stem cell (hESC)-derived NR via regulation of cell cycle progression, cytoskeleton dynamic and apical constriction of RPCs in the ciliary marginal zone (CMZ). Furthermore, activation of SCF/SCFR signaling promotes neurogenesis in the central-most NR via acceleration of the migration of immature ganglion cells and repressing apoptosis. Our study reveals an unreported role for SCF/SCFR signaling in controlling ciliary marginal cellular behaviors during early morphogenesis and neurogenesis of the human embryonic NR, providing a new potential therapeutic target for human congenital eye diseases such as anophthalmia, microphthalmia and congenital high myopia.