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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.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Blindness, associated with death of retinal cells at the back of the eye, is caused by a number of conditions with high prevalence such as glaucoma, age-related macular degeneration, and diabetic retinopathy. In addition, a large number of orphan inherited (mostly monogenic) conditions, such as retinitis pigmentosa and Leber Congenital Amaurosis, add to the overall number of patients with blinding retinal degenerative diseases. Blindness caused by deterioration and loss of retina is so far incurable. Modern biomedical research leveraging molecular and regenerative medicine approaches had a number of groundbreaking discoveries and proof-of-principle treatments of blindness in animals. However, these methods are slow to be standardized and commercialized as therapies to benefit people losing their eyesight due to retinal degenerative conditions. In this review, we will outline major regenerative medicine approaches, which are emerging as promising for preserving or/and restoring vision. We discuss the potential of each of these approaches to reach commercialization step and be converted to treatments, which could at least ameliorate blindness caused by retinal cell death.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.

PurposeTo 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.MethodsCSC14 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.ResultsRetinal 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.ConclusionsThis 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.

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.

PurposeTo create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats.MethodsFemale 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.ResultsApproximately 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.ConclusionsThis new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eμ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.

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.

PurposeTo 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).Methods3D 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.ResultsRetina 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.ConclusionsThese 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.

PurposeTo characterize a recently developed model, the retinal degenerate immunodeficient S334ter line-3 rat (SD-Foxn1 Tg(S334ter)3Lav) (RD nude rat), and to test whether transplanted rat fetal retinal sheets can elicit lost responses to light.MethodsNational Institutes of Health nude rats (SD-Foxn1 Tg) with normal retina were compared to RD nude rats with and without transplant for morphology and visual function. Retinal sheets from transgenic rats expressing human placental alkaline phosphatase (hPAP) were transplanted into the subretinal space of RD nude rats between postnatal day (P) 26 and P38. Transplant morphology was examined in vivo using optical coherence tomography (OCT). Visual function was assessed by optokinetic (OKN) testing, electroretinogram (ERG), and superior colliculus (SC) electrophysiology. Cryostat sections were analyzed for various retinal/synaptic markers and for the expression of donor hPAP.ResultsOptical coherence tomography scans showed the placement and laminar development of retinal sheet transplants in the subretinal space. Optokinetic testing demonstrated a deficit in visual acuity in RD nude rats that was improved after retinal sheet transplantation. No ERG responses were detected in the RD nude rats with or without transplantation. Superior colliculus responses were absent in age-matched control and sham surgery RD nude rats; however, robust light-evoked responses were observed in a specific location in the SC of transplanted RD nude rats. Responsive regions corresponded to the area of transplant placement in the eye. The quality of visual responses correlated with transplant organization and placement.ConclusionsThe data suggest that retinal sheet transplants integrate into the host retina of RD nude rats and recover significant visual function.

There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e., alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of > 50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicants is proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the > 100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.

The isotope ecology of great apes is a useful reference for palaeodietary reconstructions in fossil hominins. As extant apes live in C3-dominated habitats, variation in isotope signatures is assumed to be low compared to hominoids exploiting C4-plant resources. However, isotopic differences between sites and between and within individuals are poorly understood due to the lack of vegetation baseline data. In this comparative study, we included all species of free-ranging African great apes (Pan troglodytes, Pan paniscus, Gorilla sp.). First, we explore differences in isotope baselines across different habitats and whether isotopic signatures in apes can be related to feeding niches (faunivory and folivory). Secondly, we illustrate how stable isotopic variations within African ape populations compare to other extant and extinct primates and discuss possible implications for dietary flexibility. Using 701 carbon and nitrogen isotope data points resulting from 148 sectioned hair samples and an additional collection of 189 fruit samples, we compare six different great ape sites. We investigate the relationship between vegetation baselines and climatic variables, and subsequently correct great ape isotope data to a standardized plant baseline from the respective sites. We obtained temporal isotopic profiles of individual animals by sectioning hair along its growth trajectory. Isotopic signatures of great apes differed between sites, mainly as vegetation isotope baselines were correlated with site-specific climatic conditions. We show that controlling for plant isotopic characteristics at a given site is essential for faunal data interpretation. While accounting for plant baseline effects, we found distinct isotopic profiles for each great ape population. Based on evidence from habituated groups and sympatric great ape species, these differences could possibly be related to faunivory and folivory. Dietary flexibility in apes varied, but temporal variation was overall lower than in fossil hominins and extant baboons, shifting from C3 to C4-resources, providing new perspectives on comparisons between extinct and extant primates.

The cleavage motif in the hemagglutinin (HA) protein of highly pathogenic H5 and H7 subtypes of avian influenza viruses is characterized by a peptide insertion or a multibasic cleavage site (MBCS). Here, we isolated an H4N2 virus from quails (Quail/CA12) with two additional arginines in the HA cleavage site, PEKRRTR/G, forming an MBCS-like motif. Quail/CA12 is a reassortant virus with the HA and neuraminidase (NA) gene most similar to a duck-isolated H4N2 virus, PD/CA06 with a monobasic HA cleavage site. Quail/CA12 required exogenous trypsin for efficient growth in culture and caused no clinical illness in infected chickens. Quail/CA12 had high binding preference for α2,6-linked sialic acids and showed higher replication and transmission ability in chickens and quails than PD/CA06. Although the H4N2 virus remained low pathogenic, these data suggests that the acquisition of MBCS in the field is not restricted to H5 or H7 subtypes.

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.

PurposeThe goal of this study was to develop an immunodeficient rat model of retinal degeneration (RD nude rats) that will not reject transplanted human cells.MethodsSD-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.ResultsAfter 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.ConclusionsThis new rat model is useful for testing the effect of human cell transplantation on the restoration of vision without interference of immunosuppression.

Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a 'nursing' role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance - they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity. In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy.

Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40 days. Donor tissues were incubated with slow-releasing microspheres containing brain-derived neurotrophic factor or glial cell-derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome-sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l-glutamate, l-glutamine, glutathione, glycine, taurine, γ-aminobutyric acid (GABA) and DAPI (4',6-diamidino-2-phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL.

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.

PurposeTo develop three-dimensional (3D) constructs of retinal pigment epithelium (RPE) and early retina progenitor cells from human embryonic stem cells (hESCs).Methods3D tissue constructs were developed by culturing hESC-derived neural retinal progenitors in a matrix on top of hESC-derived RPE cells in a cell culture insert. An osmolarity gradient maintained the nutrition of the 3D cell constructs. Cross-sections through hESC-derived tissue constructs were characterized by immunohistochemistry for various transcription factors and cell markers.ResultshESC-derived tissue constructs expressed transcription factors characteristic of retinal development, such as pax6, Otx2, Chx10, retinal RAX; Brn3b (necessary for differentiation of retinal ganglion cells); and crx and nrl (role in photoreceptor development). Many cells expressed neuronal markers including nestin, beta-tubulin and microtubule-associated proteins.ConclusionsThis study shows for the first time that 3D early retinal progenitor tissue constructs can be derived from hESCs.

PurposeTo obtain three-dimensional images from retinal transplants in live animals and evaluate the placement and structural quality of the transplants.MethodsDonor retinal sheets were isolated from E19 fetuses of transgenic rats expressing human alkaline phosphatase (hPAP), and transplanted to the subretinal space of 19-56 days old S334ter-3 rat recipients with fast retinal degeneration (average age at surgery 32 days). A total of 143 rats were imaged 1 day to 2.8 months after surgery, using a Fourier-domain optical coherence tomography (FDOCT) system, with an axial resolution of 3.5 microm. The CCD A-line integration time was set at 200 micros for better visualization of degenerated retina. After targeting the transplant area, 139 or 199 consecutive slices were scanned. Projection images and movies of the retinal transplant area were computed and later compared with histology.ResultsOCT scans identified 137 of 141 transplants as a thickening of the degenerated retina. OCT indicated the laminar structure of the transplants and surgical defects, such as RPE/choroid damage with an accuracy rate between 83 and 99%. Three-dimensional projections showed the transplant position in the retina in relation to the optic disc. Histology of transplants by hPAP and hematoxylin-eosin staining was correlated with the OCT results.ConclusionsOptical coherence tomography is an excellent tool to image retinal layers in a live rat. This procedure helps to evaluate the placement and quality of the transplants in the living eye.

The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from embryonic day 19 rat fetuses expressing human placental alkaline phosphatase (hPAP), were transplanted to the subretinal space of 18 S334ter-3 rats with fast retinal degeneration at the age of 0.8-1.3 months. Recipients were killed at the age of 1.6-11.8 months. Frozen sections were analysed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for electron microscopy. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 12 rats at the age of 5.3-11.8 months. All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between -2.8 and -3.4 log cd/m(2). No such responses were found in age-matched S334ter-3 rats without transplants, or in those with sham surgery. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells, presumably of host origin. In conclusion, synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration.

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.

The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a "low light" intensity of 1cd/m2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects--either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs--still needs to be elucidated.

PurposeTo evaluate retinal sheet transplants in S334ter-line-3 retinal degenerate rats by comparing visual responses recorded electrophysiologically with morphology based on light and electron microscopy.MethodsS334ter-line-3 retinal degenerate rats (n = 7) received retinal sheet transplants between postnatal days 28 and 31. The donor tissue was derived from transgenic embryonic day 19 (E19) rat retinae expressing human placental alkaline phosphatase (hPAP). Fresh retinal sheets were gently transplanted into the subretinal space of the left eye with the help of a custom-made implantation tool. Selected rats (n = 5) were subjected to electrophysiologic evaluation of visual responses from the superior colliculus about 84-121 days after surgery. Transplanted eyes were processed for light microscopy (LM) and electron microscopy (EM) evaluations.ResultsAll the transplanted rats that were evaluated for visual responses in the brain showed responses to very low light stimulation (-3.42 to -2.8 log cd/m(2)) of the eye in a small area of the superior colliculus corresponding with the placement of the transplant in the host retina. Histologic evaluation showed that most of the transplants contained well-laminated areas with correct polarity in the subretinal space. Inside the transplant areas, rosettes of photoreceptors with inner and outer segments were found. In the laminated areas, the outer segments of photoreceptors were facing the host retinal pigment epithelium (RPE). Immunohistochemical evaluation of hPAP donor cells revealed areas with specific staining of the transplants in the subretinal space. Electron microscopic evaluation showed a glial demarcation membrane between the host and the transplant, however, processes originating from the transplant were observed inside the host retina.ConclusionsSheets of E19 rat retina transplanted into the subretinal space of S334ter-line-3 rats survived without immune rejection and continued to show visual function when tested after 3 months. Well-developed photoreceptors and many synapse types were seen within the transplants. hPAP staining showed a certain degree of integration between the host retina and the transplant suggesting that transplanted photoreceptors contributed to the restored light sensitivity.

The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.

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

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.

Optical coherence tomography (OCT), a non-invasive method, was used for qualitative assessment of fetal retinal sheet transplants by non-invasive imaging. Rhodopsin-mutant S334ter-line-3 rats with fast retinal degeneration (28-37-day old) were transplanted with fetal retinal sheets from embryonic day (E) 18-19 pigmented normal rats. Retinal thickness measurements from transplanted (n = 51), no surgery control (n = 8), and normal pigmented rat eyes (n = 6) were obtained using a Zeiss stratus OCT-3 scanning instrument. Frozen retinal sections were stained with hematoxylin/eosin. S334ter-line-3 rats showed significant reduction in OCT retinal thickness (p

Multi-unit visual responses to light intensities ranging from -6.46 to 0.81 logcd/m2 were recorded from the surface of the superior colliculus of dark-adapted normal pigmented and normal albino rats. Light sensitivity was significantly higher in albinos. The response onset latency was inversely proportional to the stimulus intensity. The progression of the stimulus intensity versus response onset latency curve showed a considerable difference between pigmented and albino rats. At low light levels, longer response onset latencies were recorded in pigmented rats than in albinos. This can be attributed to the transmission of rod-driven responses. The differences observed in the light response characteristics of albino rats may be indicative of their visual abnormalities.

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.

The aim of this study was to establish synapses between a transplant and a degenerated retina. To tackle this difficult task, a little-known but well-established CNS method was chosen: trans-synaptic pseudorabies virus (PRV) tracing. Sheets of E19 rat retina with or without retinal pigment epithelium (RPE) were transplanted to the subretinal space in 33 Royal College of Surgeons (RCS) and transgenic s334ter-5 rats with retinal degeneration. Several months later, PRV-BaBlu (expressing E. colibeta-galactosidase) or PRV-Bartha was injected into an area of the exposed superior colliculus (SC), topographically corresponding to the transplant placement in the retina. Twenty normal rats served as controls. After survival times of 1-5 days, retinas were examined for virus by X-gal histochemistry, immunohistochemistry and electron microscopy. In normal controls, virus was first seen in retinal ganglion cells and Müller glia after 1-1.5 days, and had spread to all retinal layers after 2-3 days. Virus-labeled cells were found in 16 of 19 transplants where the virus injection had retrogradely labeled the topographically correct transplant area of the host retina. Electron microscopically, enveloped and nonenveloped virus could clearly be detected in infected cells. Enveloped virus was found only in neurons. Infected glial cells contained only nonenveloped virus. Neurons in retinal transplants are labeled after PRV injection into the host brain, indicating synaptic connectivity between transplants and degenerated host retinas. This study provides evidence that PRV spreads in the retina as in other parts of the CNS and is useful to outline transplant-host circuitry.

PurposeTo correlate the functional outcomes with histologic findings following transplantation of fetal retinal sheets in rd mice, and to investigate the mechanisms of visual function restoration.MethodsTwenty-one postnatal day 31-38 rd/rd (C3H/HeJ) mice were transplanted in one eye with retinal sheets (1.0 x 0.4 mm) obtained from embryonic day (E) 17 enhanced-green-fluorescent protein (eGFP) mice. Five mice underwent sham surgery without insertion of tissue. Four to five weeks after transplantation, visual responses to a light flash were recorded across the superior colliculus (SC) in seven eyes of seven transplanted mice that had clear corneas and lenses, and in all five sham surgery mice. Following the SC recording, the eyes were enucleated and processed for immunohistochemistry and examined using confocal microscopy.ResultsIn three out of the seven eyes (43%), positive responses were recorded in the SC in an area topographically corresponding to the placement of the transplant in the host retina. No responses were recorded in the untreated eyes of 5-week-old and 9-week-old rd/rd mice, and in the 9-week-old sham surgery mice. In contrast, visual responses were recorded over the entire SC in normal eyes. The response onset latencies of the 3 transplanted mice with responses were similar to those of normal control mice. The organization of the graft did not appear to correlate as expected with the electrophysiology results, as eyes with well-organized, laminated grafts showed no response whereas the three light-responsive eyes had rosetted or disorganized grafts. All three light-responsive eyes demonstrated much higher levels of recoverin immunoreactivity in the host retina overlying the graft compared with untreated age-matched rd/rd mice.ConclusionRestoration of the SC visual response does not appear to depend on a well-organized transplant in the rd mouse. Increased recoverin-staining in the host retina in light-responsive animals suggested that host cone rescue was the likely mechanism of vision restoration in this transplant model.

A previously described optokinetic testing apparatus [Nat. Neurosci. 5 (2002) 53] was modified to measure vision in each eye separately for evaluation of monocular treatments. This apparatus consists also of a striped rotating drum. Ca. 170 degrees of the drum are illuminated from outside and ca. 190 degrees of the drum move behind a stationary black wall. The rat sits unrestrained in the drum center in a tube so that one eye is unexposed to the rotating stripes. Normal pigmented and retinal degenerate transgenic S334ter-3 rats were tested with the original and the modified apparatus. The usefulness of this method was tested in retinal degenerate rats with retinal transplants in one eye. In retinal degenerate animals, the amount of time (seconds) spent for head-tracking tended to be higher with the original method, possibly due to simultaneous stimulation of both eyes. In rats with retinal transplants, visual responses were significantly preserved in transplanted eyes at late stages of retinal degeneration. In conclusion, contributions from the fellow eye to the optokinetic tracking response can be limited by this testing modification, which is useful for evaluation of treatment effects to one eye.

PurposeOne of the critical difficulties in design of a high-resolution retinal implant is the proximity of stimulating electrodes to the target cells. This is a report of a phenomenon of retinal cellular migration into a perforated membrane that may help to address this problem.MethodsMylar membranes with an array of perforations (3-40 microm in diameter) were used as a substrate for in vitro retinal culture (chicken, rats) and were also transplanted into the subretinal space of adult RCS rats. A membrane was also constructed with a seal on one side to restrict the migration.ResultsRetinal tissue in vitro grew within 3 days through perforations of greater than 5 microm in diameter when the membranes were positioned on the photoreceptor side, but no migration occurred if the implant was placed on the inner retinal surface. Histology with light microscopy and transmission electron microscopy (TEM) demonstrated that migrating cells retain neuronal structures for signal transduction. Similar growth of RCS rat retinal cells occurred in vivo within 5 days of implantation. A basal seal kept the migrating tissue within a small membrane compartment.ConclusionsRetinal neurons migrate within a few days into perforations (> 5 microm in diameter) of a membrane placed into the subretinal space. This may provide a means of gaining close proximity between electrodes in a retinal prosthetic chip and target cells, and thus allow a greater density of stimulating elements to subserve higher resolution. Further studies are needed to explore the long-term stability of the retinal migration.

PurposeTo determine whether retinal transplantation can preserve visual responses in the superior colliculus (SC) of the S334ter-line-5 rat, a transgenic model for slow photoreceptor degeneration, which is more similar to human retinitis pigmentosa than the fast degeneration line 3 S334ter rat.MethodsVisual responses to a light flash were recorded in the SC. Rats that had received embryonic day (E) 19-20 fetal retinal sheet transplants at the age of 26-30 days were tested at the ages of 200-254 days. Controls were age-matched rats without surgery and with sham surgery. As a baseline, in no-surgery line-5 rats, the temporal pattern of visual sensitivity loss was evaluated electrophysiologically in the SC from 60 days up to one year of age.ResultsIn untreated S334ter-line-5 rats, decline in visual sensitivity in the SC was parallel to the photoreceptor loss. At 109 day of age, a relative scotoma developed in the area of the SC corresponding to the nasal retinal region. At 200-254 days of age, the majority of the SC was devoid of any light-driven responses. In contrast, at this time point, transplanted rats with 'good' retinal grafts with normal lamination had visual responses in the caudal region of the SC, the area corresponding topographically to the transplant location in the retina. In these rats, the various parameters of SC responses such as the latency of the onset of the visual response, the response peak amplitude and the consistency of the visual response were significantly different from the control groups (no-surgery, sham surgery, 'poor' transplants) and were more comparable to normal albino rats, however, with a slightly longer latency (70-90 vs. 30-50 msec).ConclusionsFetal retinal sheet transplantation showed a long-term rescue effect on visual function in this animal model of slow photoreceptor degeneration.

PurposeTo investigate different in vitro model systems for retinal progenitor cell (RPC) isolation and expansion.MethodsRPCs were isolated from embryonic day (E) 17 Long Evans rat retinas. Three different culture media: (1) modified serum free defined media (2) serum-containing media and (3) embryonic stem cell (ES)-conditioned media were used for RPC isolation and long term expansion. Expression of various cellular markers and cell morphologies were compared among the three culture systems at different passages by immunostaining and confocal microscopy.ResultsAll three culture systems could maintain RPCs as nestin-positive cells (78-87%) after long-term in vitro expansion. However, RPCs appeared to proliferate faster in the serum-free culture system. The ES-conditioned media provided the best RPC survival. Cells appeared smaller at early passages compared with later passages. This morphology change occurred at P9-P10 in the serum-free medium, and at P5-P6 in the other two culture systems.ConclusionsThe serum-free medium may be superior for preventing RPC differentiation during expansion.

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

PURPOSE:To map the spatiotemporal decline in retinally driven activity in the superior colliculus (SC) of transgenic S334ter-line-3 rats that express a mutated rhodopsin, which causes photoreceptor degeneration. To determine whether transplantation of fetal retinal sheets into the subretinal space of these rats can recover visual activity in the SC. METHODS:A visual stimulus was presented to the eye, and responses were recorded across the SC of untreated S334ter-line-3 rats aged 28 to 288 days. These data were used to draw a map of the developing scotoma. Intact retinal sheets from embryonic day 19 rats were transplanted into the subretinal space of S334ter-line-3 rats between 21 and 28 days of age. Responses to retinal stimulation were mapped in the SC of transplanted and sham control rats 78 to 163 days after surgery. The morphology of the retinas in all groups was examined. RESULTS:Photoreceptor cell loss in untreated rats matched the decline in visual activity in the SC. At 28 days, there was a scotoma in the area of the SC that represents the central retina and, by 63 days, it had enlarged to cover the entire retinal representation. Visual responses were evoked in 64% of rats with retinal transplants. These retinally driven responses were confined to a small, contiguous region of the SC that represents the sector of the retina where the transplant was placed. Visual responses were absent in the SC outside this area in transplant recipients and throughout the SC of untreated and sham control rats. CONCLUSIONS:Transplantation of fetal retinal sheets induced recovery of visual activity in the SC in this model of RP. The mechanisms underlying this functional recovery remain to be resolved, but these results suggest that transplantation should be further explored as a therapy for RP.

To identify and characterize the lineage potential of rat neural retina progenitor cells (NRPCs) in vitro and engrafted into rats with retinal degeneration, NRPCs were isolated from neural retinas of embryonic day 17 Long Evans rats and cultured in serum-free or serum-containing media with fibroblast growth factor 2 and neurotrophin 3. After expansion, cellular differentiation was initiated by the withdrawal of these growth factors. Despite forming primary neurospheres, NRPCs cultured in serum-free medium survived poorly after passage. In contrast, NRPCs cultured in serum-containing medium could be expanded for up to 12 passages and differentiated into glial fibrillary acidic protein-positive glial cells and retina-specific neurons expressing rhodopsin, S-antigen, calbindin, recoverin, and calretinin. For in vivo analysis, passage 1 (P1) undifferentiated NRPCs were labeled with bromodeoxyuridine (BrdU), implanted into the subretinal space of Royal College of Surgeons (RCS) rats, and analyzed immunohistochemically 4 weeks postgrafting. The grafted NRPCs showed extensive glial differentiation, irrespective of their topographic localization. A few BrdU-labeled grafted NRPCs expressed protein kinase C, a marker for bipolar and amacrine interneuron-specific differentiation. Other retina-specific or oligodendrocytic differentiation was not detected in the grafted cells. Although NRPCs are capable of self-renewal and multilineage differentiation in vitro, they developed mostly into glial cells following engraftment into the adult retina. These data suggest that the adult retina retains epigenetic signals that are either restrictive for neuronal differentiation or instructive for glial differentiation. Induction of lineage-specific cell differentiation of engrafted NRPCs to facilitate retinal repair will likely require initiation of specific differentiation in vitro prior to grafting and/or modification of the host environment concomitantly with NRPC grafting.

This study investigated whether transplanted sheets of human fetal retina together with its retinal pigment epithelium (RPE) could develop and maintain their cytoarchitecture after long survival times. Transplant recipients were nine albino athymic nu/nu rats with a normal retina. The donor tissue was dissected from fetuses of 12-17 weeks gestational age. Transplants were analyzed at 5-12 months after surgery by light and electron microscopy, and immunohistochemistry with various antibodies specific for rhodopsin, S-antigen, transducin, neurofilament and synaptophysin. In 4 of 11 transplants, the RPE stayed as a monolayer sheet and supported the development of the retinal sheet with a normal lamination, including photoreceptor inner and outer segments. Cones and rods in the organized transplants were labeled with different photoreceptor markers. Inner and outer plexiform layers, containing cone pedicles and rods spherules, were immunoreactive for synaptophysin. As the recipients had a normal retina, transplant/host integration was not expected. However, at the transplant/host interface, there were sometimes areas without glial barriers, and neurofilament-containing processes could be observed crossing between transplant and host. In other, more disorganized transplants, the RPE cells were partially dispersed or clumped together in clusters. Such transplants developed photoreceptors in rosettes, often with inner and outer segments. In conclusion, sheets of human fetal retina transplanted together with its RPE to the subretinal space of nude rats can develop and maintain perfectly laminated transplants after long survival times, indicating the potential of applying cotransplantation to human patients with retinal diseases.

PurposeTo assess whether transplantation of intact sheets of fetal retina with retinal pigment epithelium (RPE) into a retina with photoreceptor degeneration restores visually evoked responses.MethodsSheets of fetal retina with RPE were transplanted into the subretinal space of Royal College of Surgeons (RCS) rats at 37 to 69 days of age. Sixty-three days to 10 months after transplantation, multiunit visual responses were recorded in the superior colliculus (SC) of transplanted rats, age-matched untransplanted rats, and rats with sham surgery.ResultsIn 19 of 29 RCS rats with transplants, visually evoked responses were recorded from and restricted to a small area of the SC that corresponds topographically to the portion of the retina in which the transplant was placed. Outside of this area, no visual responses were evoked. Visually evoked responses were never recorded in age-matched, nontransplanted RCS rats. Visually evoked responses were recorded in 6 of 13 RCS rats with sham surgery, but these responses were significantly different from responses in rats with transplants.ConclusionsThese results demonstrate that this transplantation technique restores visually evoked responses in the brain. Although the underlying mechanism is unknown, we propose that the central visual response results from increased synaptic efficacy within the host retina. If it can be established that functional connections between the transplant and the host retina produce the effect, then it would indicate that the technique could be explored as a therapeutic strategy in some diseases of retinal degeneration.

PurposeTo develop a retinal degeneration model with selective photoreceptor loss and RPE sparing, to be used as recipient for evaluating retinal transplants.MethodsAlbino rats were exposed to blue light, continuously, for 1-7 days (24-168 h) in a specially designed cage. Eyes were histologically analyzed at periods between 2 h and 8 months after the light exposure. Electroretinograms (ERGs) were recorded from some rats at 12-216 days after exposure. Using behavioral methods, visual thresholds of some rats were determined before exposure and re-measured between 18 and 52 days following exposure.ResultsApoptotic nuclei appeared exclusively in the photoreceptor layer after 1-5 days exposure to blue light. Light microscopy revealed that 2-4 days of light exposure reduced the outer nuclear layer (normally eight to ten rows) to 1 row of cells in the central retina and to two to three rows in the periphery, both in the superior and the inferior retina. Average ERG a- and b-wave amplitudes of light-damaged rats were both reduced by about 98%. Visual performance in the behavioral test was substantially impaired.ConclusionsContinuous exposure of albino rats to moderate blue light for 2-5 days selectively eliminates most of the photoreceptors while leaving the RPE initially intact.

The aim was to demonstrate functional properties of transplanted histologically normal photoreceptors. Subretinal intact-sheet transplants of fetal E17-E20 rat retinas to light-damaged albino rat eyes were fixed in light or dark, 2 to 42 weeks after transplantation, and stained immunohistochemically for certain phototransduction proteins. In light adapted transplants, transducin was predominantly found in inner segments of parallel-organized photoreceptors. Transducin shifted to the outer segments with dark-adaptation. S-antigen distribution was opposite to transducin. Rhodopsin distribution did not change. The shift of signal transduction proteins correlated to the light conditions indicates that normal phototransduction processes were established in photoreceptors of transplanted retinal sheets.

The idea of implanting microphotodiode arrays as visual prostheses has aroused controversy on its feasibility from the moment it appeared in print. We now present results which basically support the concept of replacing damaged photoreceptors with subretinally implanted stimulation devices. Network activity in degenerated rat retinae could be modulated through local electrical stimulation in vitro. We also investigated the long term stability and biocompatibility of the subretinal implants and their impact on retinal physiology in rats. Ganzfeld electroretinograms and histology showed no significant side effect of subretinal implants on retinal function or the architecture of the inner retina.

PurposeMany retinal diseases, such as macular degeneration, affect both retinal pigment epithelium (RPE) and photoreceptors. Therefore, retinal repair may require transplantation of both tissues together as a cograft.MethodsAs recipients of retina-RPE cografts, 7- to 10-week-old albino Royal College of Surgeons rats that lose their photoreceptors because of a pigment epithelium defect were used. Freshly harvested intact sheets of RPE with neural retina from pigmented normal rat fetuses were gel embedded for protection and transplanted into the subretinal space.ResultsAfter 6 to 7 weeks, with the support of the cografted RPE sheet, transplanted photoreceptors developed fully in organized parallel layers in the subretinal space. Immunohistochemistry for rhodopsin, rod alpha-transducin, and S-antigen and peanut agglutinin labeling for cone interphotoreceptor matrix domains suggested that the photoreceptors in the graft were capable of normal function.ConclusionsFreshly harvested intact sheets of fetal RPE and retina, transplanted together into the subretinal space, can develop a normal morphology. Such transplants have the potential to benefit retinal diseases with dysfunctional RPE and photoreceptors.

PurposeThe aim of this study was to establish a model for morphologic retinal reconstruction after destruction of photoreceptors.MethodsRat embryos were prelabeled by injection of bromodeoxyuridine (BrdU) into timed pregnant rats on 2 to 6 consecutive days. Pieces of fetal retinas (embryonic day [E] 17 to E22) were embedded in growth factor-reduced matrigel for protection and stored in medium on ice. With the use of a custom-mnade implantation tool, trimmed embedded pieces were placed into the subretinal space of albino rats whose photoreceptors had been damaged by continuous exposure to blue light for 3 to 4 days.ResultsDonor cells were unequivocally identified by the BrdU label. Approximately 25% of transplants in the subretinal space developed parallel layers, with photoreceptor outer segments facing the host pigment epithelium. Transplants developed rosettes if host pigment epithelium had been damaged, if trauma to the donor tissue occurred during preparation or transplantation, and if the donor tissue was misplaced into the choroid or into the epiretinal space on top of the host retina. If the surgery was performed more than 4 weeks after the light damage, continued degeneration of the host retina caused secondary pigment epithelium damage, and transplants did not develop parallel layers of photoreceptor outer segments.ConclusionsAfter transplantation to the subretinal space of a degenerated retina, gel-protected fetal retina can develop parallel layers and photoreceptor outer segments in contact with host pigment epithelium. Transplants can develop good fusion with the inner retina of a photoreceptor-deficient recipient.