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.

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.

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.

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.

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.

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.

Organoid-derived C-Kit+/SSEA4- human retinal progenitor cells promote a protective retinal microenvironment during transplantation in rodents.

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.

Validation and Safety of Visual Restoration by Ectopic Expression of Human Melanopsin in Retinal Ganglion Cells.

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.

Investigating oxidation state-induced toxicity of PEGylated graphene oxide in ocular tissue using gene expression profiles.

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.

Bone Marrow CD133+ Stem Cells Ameliorate Visual Dysfunction in Streptozotocin-induced Diabetic Mice with Early Diabetic Retinopathy.

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.

Long-term safety of human retinal progenitor cell transplantation in retinitis pigmentosa patients.

BACKGROUND: Retinitis pigmentosa is a common genetic disease that causes retinal degeneration and blindness for which there is currently no curable treatment available. Vision preservation was observed in retinitis pigmentosa animal models after retinal stem cell transplantation. However, long-term safety studies and visual assessment have not been thoroughly tested in retinitis pigmentosa patients. METHODS: In our pre-clinical study, purified human fetal-derived retinal progenitor cells (RPCs) were transplanted into the diseased retina of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. Based on these results, we conducted a phase I clinical trial to establish the safety and tolerability of transplantation of RPCs in eight patients with advanced retinitis pigmentosa. Patients were studied for 24 months. RESULTS: After RPC transplantation in RCS rats, we observed moderate recovery of vision and maintenance of the outer nuclear layer thickness. Most importantly, we did not find tumor formation or immune rejection. In the retinis pigmentosa patients given RPC injections, we also did not observe immunological rejection or tumorigenesis when immunosuppressive agents were not administered. We observed a significant improvement in visual acuity (P < 0.05) in five patients and an increase in retinal sensitivity of pupillary responses in three of the eight patients between 2 and 6 months after the transplant, but this improvement did not appear by 12 months. CONCLUSION: Our study for the first time confirmed the long-term safety and feasibility of vision repair by stem cell therapy in patients blinded by retinitis pigmentosa. TRIAL REGISTRATION: WHO Trial Registration, ChiCTR-TNRC-08000193 . Retrospectively registered on 5 December 2008.

Evidence for a retinal progenitor cell in the postnatal and adult mouse.

Progress in cell therapy for retinal disorders has been challenging. Recognized retinal progenitors are a heterogeneous population of cells that lack surface markers for the isolation of live cells for clinical implementation. In the present application, our objective was to use the stem cell factor receptor c-Kit (CD117), a surface marker, to isolate and evaluate a distinct progenitor cell population from retinas of postnatal and adult mice. Here we report that, by combining traditional methods with fate mapping, we have identified a c-Kit-positive (c-Kit+) retinal progenitor cell (RPC) that is self-renewing and clonogenic in vitro, and capable of generating many cell types in vitro and in vivo. Based on cell lineage tracing, significant subpopulations of photoreceptors in the outer nuclear layer and bipolar, horizontal, amacrine and Müller cells in the inner nuclear layer are the progeny of c-Kit+ cells in vivo. The RPC progeny contributes to retinal neurons and glial cells, which are responsible for the conversion of light into visual signals. The ability to isolate and expand in vitro live c-Kit+ RPCs makes them a future therapeutic option for retinal diseases.