Detailed Visual Cortical Responses Generated by Retinal Sheet Transplants in Rats with Severe Retinal Degeneration.

To combat retinal degeneration, healthy fetal retinal sheets have been successfully transplanted into both rodent models and humans, with synaptic connectivity between transplant and degenerated host retina having been confirmed. In rodent studies, transplants have been shown to restore responses to flashes of light in a region of the superior colliculus corresponding to the location of the transplant in the host retina. To determine the quality and detail of visual information provided by the transplant, visual responsivity was studied here at the level of visual cortex where higher visual perception is processed. For our model, we used the transgenic Rho-S334ter line-3 rat (both sexes), which loses photoreceptors at an early age and is effectively blind at postnatal day 30. These rats received fetal retinal sheet transplants in one eye between 24 and 40 d of age. Three to 10 months following surgery, visually responsive neurons were found in regions of primary visual cortex matching the transplanted region of the retina that were as highly selective as normal rat to stimulus orientation, size, contrast, and spatial and temporal frequencies. Conversely, we found that selective response properties were largely absent in nontransplanted line-3 rats. Our data show that fetal retinal sheet transplants can result in remarkably normal visual function in visual cortex of rats with a degenerated host retina and represents a critical step toward developing an effective remedy for the visually impaired human population.SIGNIFICANCE STATEMENT Age-related macular degeneration and retinitis pigmentosa lead to profound vision loss in millions of people worldwide. Many patients lose both retinal pigment epithelium and photoreceptors. Hence, there is a great demand for the development of efficient techniques that allow for long-term vision restoration. In this study, we transplanted dissected fetal retinal sheets, which can differentiate into photoreceptors and integrate with the host retina of rats with severe retinal degeneration. Remarkably, we show that transplants generated visual responses in cortex similar in quality to normal rats. Furthermore, transplants preserved connectivity within visual cortex and the retinal relay from the lateral geniculate nucleus to visual cortex, supporting their potential application in curing vision loss associated with retinal degeneration.

Sheets of human retinal progenitor transplants improve vision in rats with severe retinal degeneration.

Loss of photoreceptors and other retinal cells is a common endpoint in retinal degenerate (RD) diseases that cause blindness. Retinal transplantation is a potential therapy to replace damaged retinal cells and improve vision. In this study, we examined the development of human fetal retinal sheets with or without their retinal pigment epithelium (RPE) transplanted to immunodeficient retinal degenerate rho S334ter-3 rats. Sheets were dissected from fetal human eyes (11-15.7 weeks gestation) and then transplanted to the subretinal space of 24-31 d old RD nude rats. Every month post surgery, eyes were imaged by high-resolution spectral-domain optical coherence tomography (SD-OCT). SD-OCT showed that transplants were placed into the subretinal space and developed laminated areas or rosettes, with clear development of plexiform layers first seen in OCT at 3 months post surgery. Several months later, as could be expected by the much slower development of human cells compared to rat cells, transplant photoreceptors developed inner and later outer segments. Retinal sections were analyzed by immunohistochemistry for human and retinal markers and confirmed the formation of several retinal subtypes within the retinal layers. Transplant cells extended processes and a lot of the cells could also be seen migrating into the host retina. At 5.8-8.6 months post surgery, selected rats were exposed to light flashes and recorded for visual responses in superior colliculus, (visual center in midbrain). Four of seven rats with transplants showed responses to flashes of light in a limited area of superior colliculus. No response with the same dim light intensity was found in age-matched RD controls (non-surgery or sham surgery). In summary, our data showed that human fetal retinal sheets transplanted to the severely disturbed subretinal space of RD nude rats develop mature photoreceptors and other retinal cells, integrate with the host and induce vision improvement.

A new immunodeficient retinal dystrophic rat model for transplantation studies using human-derived cells.

PURPOSE: To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats. METHODS: Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control. RESULTS: Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection. CONCLUSIONS: This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

A new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression.

PURPOSE: The goal of this study was to develop an immunodeficient rat model of retinal degeneration (RD nude rats) that will not reject transplanted human cells. METHODS: SD-Tg(S334ter)3Lav females homozygous for a mutated mouse rhodopsin transgene were mated with NTac:NIH-Whn (NIH nude) males homozygous for the Foxn1 (rnu) allele. Through selective breeding, a new stock, SD-Foxn1 Tg(S334ter)3Lav (RD nude) was generated such that all animals were homozygous for the Foxn1 (rnu) allele and either homo- or hemizygous for the S334ter transgene. PCR-based assays for both the Foxn1 (rnu) mutation and the S334ter transgene were developed for accurate genotyping. Immunodeficiency was tested by transplanting sheets of hESC-derived neural progenitor cells to the subretinal space of RD nude rats, and, as a control, NIH nude rats. Rats were killed between 8 and 184 days after surgery, and eye sections were analyzed for human, neuronal, and glial markers. RESULTS: After transplantation to RD nude and to NIH nude rats, hESC-derived neural progenitor cells differentiated to neuronal and glial cells, and migrated extensively from the transplant sheets throughout the host retina. Migration was more extensive in RD nude than in NIH nude rats. Already 8 days after transplantation, donor neuronal processes were found in the host inner plexiform layer. In addition, host glial cells extended processes into the transplants. The host retina showed the same photoreceptor degeneration pattern as in the immunocompetent SD-Tg(S334ter)3Lav rats. Recipients survived well after surgery. CONCLUSIONS: This new rat model is useful for testing the effect of human cell transplantation on the restoration of vision without interference of immunosuppression.

Survival and Functional Integration of Human Embryonic Stem Cell-Derived Retinal Organoids After Shipping and Transplantation into Retinal Degeneration Rats.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

The Prospects for Retinal Organoids in Treatment of Retinal Diseases.

Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which RO-derived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.

Deep Learning-Assisted Multiphoton Microscopy to Reduce Light Exposure and Expedite Imaging in Tissues With High and Low Light Sensitivity.

Purpose: Two-photon excitation fluorescence (2PEF) reveals information about tissue function. Concerns for phototoxicity demand lower light exposure during imaging. Reducing excitation light reduces the quality of the image by limiting fluorescence emission. We applied deep learning (DL) super-resolution techniques to images acquired from low light exposure to yield high-resolution images of retinal and skin tissues. Methods: We analyzed two methods: a method based on U-Net and a patch-based regression method using paired images of skin (550) and retina (1200), each with low- and high-resolution paired images. The retina dataset was acquired at low and high laser powers from retinal organoids, and the skin dataset was obtained from averaging 7 to 15 frames or 70 frames. Mean squared error (MSE) and the structural similarity index measure (SSIM) were outcome measures for DL algorithm performance. Results: For the skin dataset, the patches method achieved a lower MSE (3.768) compared with U-Net (4.032) and a high SSIM (0.824) compared with U-Net (0.783). For the retinal dataset, the patches method achieved an average MSE of 27,611 compared with 146,855 for the U-Net method and an average SSIM of 0.636 compared with 0.607 for the U-Net method. The patches method was slower (303 seconds) than the U-Net method (<1 second). Conclusions: DL can reduce excitation light exposure in 2PEF imaging while preserving image quality metrics. Translational Relevance: DL methods will aid in translating 2PEF imaging from benchtop systems to in vivo imaging of light-sensitive tissues such as the retina.

Tissue Engineering Strategies for Retina Regeneration.

The retina is a complex and fragile photosensitive part of the central nervous system which is prone to degenerative diseases leading to permanent vision loss. No proven treatment strategies exist to treat or reverse the degenerative conditions. Recent investigations demonstrate that cell transplantation therapies to replace the dysfunctional retinal pigment epithelial (RPE) cells and or the degenerating photoreceptors (PRs) are viable options to restore vision. Pluripotent stem cells, retinal progenitor cells, and somatic stem cells are the main cell sources used for cell transplantation therapies. The success of retinal transplantation based on cell suspension injection is hindered by limited cell survival and lack of cellular integration. Recent advances in material science helped to develop strategies to grow cells as intact monolayers or as sheets on biomaterial scaffolds for transplantation into the eyes. Such implants are found to be more promising than the bolus injection approach. Tissue engineering techniques are specifically designed to construct biodegradable or non-degradable polymer scaffolds to grow cells as a monolayer and construct implantable grafts. The engineered cell construct along with the extracellular matrix formed, can hold the cells in place to enable easy survival, better integration, and improved visual function. This article reviews the advances in the use of scaffolds for transplantation studies in animal models and their application in current clinical trials.

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats.

End-stage age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two major retinal degenerative (RD) conditions that result in irreversible vision loss. Permanent eye damage can also occur in battlefields or due to accidents. This suggests there is an unmet need for developing effective strategies for treating permanent retinal damages. In previous studies, co-grafted sheets of fetal retina with its retinal pigment epithelium (RPE) have demonstrated vision improvement in rat retinal disease models and in patients, but this has not yet been attempted with stem-cell derived tissue. Here we demonstrate a cellular therapy for irreversible retinal eye injuries using a "total retina patch" consisting of retinal photoreceptor progenitor sheets and healthy RPE cells on an artificial Bruch's membrane (BM). For this, retina organoids (ROs) (cultured in suspension) and polarized RPE sheets (cultured on an ultrathin parylene substrate) were made into a co-graft using bio-adhesives [gelatin, growth factor-reduced matrigel, and medium viscosity (MVG) alginate]. In vivo transplantation experiments were conducted in immunodeficient Royal College of Surgeons (RCS) rats at advanced stages of retinal degeneration. Structural reconstruction of the severely damaged retina was observed based on histological assessments and optical coherence tomography (OCT) imaging. Visual functional assessments were conducted by optokinetic behavioral testing and superior colliculus electrophysiology. Long-term survival of the co-graft in the rat subretinal space and improvement in visual function were observed. Immunohistochemistry showed that co-grafts grew, generated new photoreceptors and developed neuronal processes that were integrated into the host retina. This novel approach can be considered as a new therapy for complete replacement of a degenerated retina.

Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance.

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy.

Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Trophic factors GDNF and BDNF improve function of retinal sheet transplants.

The aim of this study was to compare glial-derived neurotrophic factor (GDNF) treatment with brain-derived neurotrophic factor (BDNF) treatment of retinal transplants on restoration of visual responses in the superior colliculus (SC) of the S334ter line 3 rat model of rapid retinal degeneration (RD). RD rats (age 4-6 weeks) received subretinal transplants of intact sheets of fetal retina expressing the marker human placental alkaline phosphatase (hPAP). Experimental groups included: (1) untreated retinal sheet transplants, (2) GDNF-treated transplants, (3) BDNF-treated transplants, (4) none surgical, age-matched RD rats, (5) sham surgery RD controls, (6) progenitor cortex transplant RD controls, and (7) normal pigmented rat controls. At 2-8 months after transplantation, multi-unit visual responses were recorded from the SC using a 40 ms full-field stimulus (-5.9 to +1 log cd/m(2)) after overnight dark-adaptation. Responses were analyzed for light thresholds, spike counts, response latencies, and location within the SC. Transplants were grouped into laminated or rosetted (more disorganized) transplants based on histological analysis. Visual stimulation of control RD rats evoked no responses. In RD rats with retinal transplants, a small area of the SC corresponding to the position of the transplant in the host retina, responded to light stimulation between -4.5 and -0.08 log cd/m(2), whereas the light threshold of normal rats was at or below -5 log cd/m(2) all over the SC. Overall, responses in the SC in rats with laminated transplants had lower response thresholds and were distributed over a wider area than rats with rosetted transplants. BDNF treatment improved responses (spike counts, light thresholds and responsive areas) of rats with laminated transplants whereas GDNF treatment improved responses from rats with both laminated and rosetted (more disorganized) transplants. In conclusion, treatment of retinal transplants with GDNF and BDNF improved the restoration of visual responses in RD rats; and GDNF appears to exert greater overall restoration than BDNF.

Retina Organoid Transplants Develop Photoreceptors and Improve Visual Function in RCS Rats With RPE Dysfunction.

Purpose: To study if human embryonic stem cell-derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction. Methods: CSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30-70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers. Results: Retinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6-8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host. Conclusions: This study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Functional and structural assessment of retinal sheet allograft transplantation in feline hereditary retinal degeneration.

PURPOSE: To investigate whether sheets of fetal retinal allografts can integrate into the dystrophic Abyssinian cat retina with progressive rod cone degeneration. METHODS: Fetal retinal sheets (cat gestational day 42), incubated with BDNF microspheres, were transplanted to the subretinal space of four cats at an early disease stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and indocyanine green and fluorescein angiograms up to 4 months following surgery. E42 donor and transplanted eyes were analyzed by histology and immunohistochemistry for retinal markers. RESULTS: Funduscopy and angiography showed good integration of the transplants in two of four cats, including extension of host blood vessels into the transplant and some scarring in the host. In these two, transplants were found in the subretinal space with laminated areas, with photoreceptor outer segments in normal contacts with the host retinal pigment epithelium. In some areas, transplants appeared to be well-integrated within the host neural retina. Neither of these two cats showed functional improvement in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants stained for all investigated cellular markers. No PKC immunoreactivity was detected in the fetal donor retina at E42, but developed in the 4-month-old grafts. CONCLUSIONS: Fetal sheet transplants can integrate well within a degenerating cat retina and develop good lamination of photoreceptors. Functional improvement was not demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further evaluated in cat host retinas with a more advanced retinal degeneration using longer follow-up times.

Retinal transplants restore visual responses: trans-synaptic tracing from visually responsive sites labels transplant neurons.

This study aimed to test the hypothesis that visual responses in the superior colliculus (SC) originate from synaptic connections between fetal retinal transplants and degenerating host retinas. Sheets of embryonic day 19 rat retina expressing human placental alkaline phosphatase were transplanted to the subretinal space of 3- to 4-week-old S334ter-line-3 rats with fast retinal degeneration. Several months later, visual responses were recorded from the SC. Attenuated pseudorabies virus that is specifically transferred between neurons at synapses (strains PRV-152, expressing green fluorescent protein (GFP) or BaBlu, expressing Escherichia colibeta-galactosidase) was injected into the visually responsive site of the SC. After survival times of 1-2 days, the virus was detected in the retina by immunohistochemistry in combination with different retinal cell markers, such as protein kinase C, recoverin, calcium-calmodulin-dependent protein kinase II and glutamine synthetase. Transplanted rats had a mean response threshold of -3.1 log cd/m(2) in a small area of the SC corresponding to the location of the graft in the retina. By 30 h after injection into this SC area, the virus traced back to host ganglion cells overlying the transplant and in close proximity to the transplant. By 2 days after injection, extensive virus label was found in the host retina and many cells in the transplant were also labeled. Virus-labeled cells in the transplant were double labeled for neuronal and glial cell markers. This study provides anatomical evidence that synaptic connections between fetal retinal transplants and host retinas contribute to the visual responses in the SC.

Expression of ZnT and ZIP zinc transporters in the human RPE and their regulation by neurotrophic factors.

PURPOSE: Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined. METHODS: Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn(2+) uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined. RESULTS: Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn(2+) to cultures resulted in a dose-dependent increase in intracellular Zn(2+) content in ARPE19, and >30 nM Zn(2+) induced necrosis with an LC(50) of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn(2+) uptake in the RPE. CONCLUSIONS: The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases.

Vision improvement in retinal degeneration patients by implantation of retina together with retinal pigment epithelium.

PURPOSE: To demonstrate efficacy and safety of the implantation of neural retinal progenitor cell layers (sheets) with its retinal pigment epithelium (RPE) in retinitis pigmentosa (RP) and dry age-related macular degeneration (AMD) patients with 20/200 or worse vision in the surgery eye. DESIGN: Interventional nonrandomized clinical trial. METHODS: Ten patients (six RP, four AMD) received retinal implants in one eye and were followed in a phase II trial conducted in a clinical practice setting. Early Treatment Diabetic Retinopathy Study (EDTRS) was the primary outcome measure. All implant recipients and nine of 10 tissue donors were deoxyribonucleic acids typed. RESULTS: Seven patients (three RP, four AMD) showed improved EDTRS visual acuity (VA) scores. Three of these patients (one RP, two AMD) showed improvement in both eyes to the same extent. Vision in one RP patient remained the same, while vision in two RP patients decreased. One RP patient has maintained an improvement in vision from 20/800 to 20/200 ETDRS for more than five years; at the six-year examination, it was still maintained at 20/320 while the nonsurgery eye had deteriorated to hand motion vision. This patient also showed a 22.72% increase in light sensitivity at five years compared to microperimetry results at two years; the other patients showed no improved sensitivity. Although no match was found between donors and recipients, no rejection of the implanted tissue was observed clinically. CONCLUSIONS: Seven (70%) of 10 patients showed improved VA. This outcome provides clinical evidence of the safety and beneficial effect of retinal implants and corroborates results in animal models of retinal degeneration.

Transplanted hESC-Derived Retina Organoid Sheets Differentiate, Integrate, and Improve Visual Function in Retinal Degenerate Rats.

Purpose: To investigate whether sheets of retina organoids derived from human embryonic stem cells (hESCs) can differentiate, integrate, and improve visual function in an immunodeficient rat model of severe retinal degeneration (RD). Methods: 3D hESC-derived retina organoids were analyzed by quantitative PCR and immunofluorescence. Sheets dissected from retina organoids (30-65 days of differentiation) were transplanted into the subretinal space of immunodeficient rho S334ter-3 rats. Visual function was tested by optokinetic testing and electrophysiologic recording in the superior colliculus. Transplants were analyzed at 54 to 300 days postsurgery by immunohistochemistry for donor and retinal markers. Results: Retina organoids contained multiple retinal cell types, including progenitor populations capable of developing new cones and rods. After transplantation into an immunodeficient rat model of severe RD, the transplanted sheets differentiated, integrated, and produced functional photoreceptors and other retinal cells, according to the longer human developmental timetable. Maturation of the transplanted retinal cells created visual improvements that were measured by optokinetic testing and electrophysiologic recording in the superior colliculus. Immunohistochemistry analysis indicated that the donor cells were synaptically active. Extensive transplant projections could be seen within the host RD retina. Optical coherence tomography imaging monitored long-term transplant growth and survival up to 10 months postsurgery. Conclusions: These data demonstrate that the transplantation of sheets dissected from hESC-derived retina organoids is a potential therapeutic method for restoring vision in advanced stages of RD.

Behavioral evaluation of visual function of rats using a visual discrimination apparatus.

A visual discrimination apparatus was developed to evaluate the visual sensitivity of normal pigmented rats (n=13) and S334ter-line-3 retinal degenerate (RD) rats (n=15). The apparatus is a modified Y maze consisting of two chambers leading to the rats' home cage. Rats were trained to find a one-way exit door leading into their home cage, based on distinguishing between two different visual alternatives (either a dark background or black and white stripes at varying luminance levels) which were randomly displayed on the back of each chamber. Within 2 weeks of training, all rats were able to distinguish between these two visual patterns. The discrimination threshold of normal pigmented rats was a luminance level of -5.37+/-0.05 log cd/m(2); whereas the threshold level of 100-day-old RD rats was -1.14+/-0.09 log cd/m(2) with considerable variability in performance. When tested at a later age (about 150 days), the threshold level of RD rats was significantly increased (-0.82+/-0.09 log cd/m(2), p<0.03, paired t-test). This apparatus could be useful to train rats at a very early age to distinguish between two different visual stimuli and may be effective for visual functional evaluations following therapeutic interventions.

Visual functional effects of constant blue light in a retinal degenerate rat model.

Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line-3 rat pups (21 days old) were exposed for 5-6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S-antigen, red-green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree(-1) in 38-day-old line 3 rats. With a 0.125 cycles degree(-1) stimulus, the head tracking performance of 80-day-old BL rats were similar to that of 220-day-old no-BL-treated line-3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light-damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.