Retinal Transplantation Research Articles

Retinal transplantation of neural progenitor cells derived from the brain of GFP transgenic mice

Neural progenitor cells isolated from the brains of neonatal GFP transgenic mice were grafted to the retina of RCS rats and rds and B6 mice. Expression of GFP and differentiation markers was evaluated at 1–4 weeks post-transplantation. Grafted cells maintained transgene expression throughout the 4-week period. At 1 week there was widespread migration of GFP + cells within the host retina and at 2 weeks evidence of neuronal differentiation (as shown by both marker expression and cell morphology), although integration at 4 weeks was limited to syngeneic recipients. Because brain-derived neural progenitor cells exhibit both neuronal and astrocytic differentiation in diseased and normal host retina, these cells provide a useful tool for studies of retinal regeneration.

Ultrastructure of adult rd mouse retina.

To examine the ultrastructure of the adult rd mouse retina in order to determine what structures are altered or lost and thus to better interpret changes produced by photoreceptor and/or retinal transplantation in this model of retinal degeneration. rd mutant mice expressing a LacZ reporter gene in rod bipolars were used in order to identify these cells and their processes. Mice of age 6 weeks to 5 months were studied by electron microscopy, concentrating on the posterior pole where retinal transplants are usually placed. The adult rd mouse retina contains degenerating cones, cone outer segments, cone synaptic pedicles with synaptic vesicles and post-synaptic contacts. The major abnormalities occur in the subretinal space where all traces of rods are gone and the main structures are inner segments of cones. These inner segments are smaller than normal, contain fewer and smaller mitochondria, have organized arrays of microtubules, resembling those in cone axonal processes, and are completely engulfed by massive proliferation of apical processes of the retinal epithelium. The subretinal space is well defined by the external limiting membrane vitreally and the retinal epithelium choroidally. Muller cells extend globular rather than filamentous processes into the subretinal space which contact the apical processes of the epithelium. Rod bipolar cells survive and retain processes in the external plexiform layer. The adult rd mouse retains structural elements necessary for phototransduction and transmission of signals to the inner layers of the retina by the cone system. The major deficits are located in the subretinal space where all rods are lost and cone inner segments undergo a slow degeneration. Rod bipolar cells survive but appear to be de-afferented; there was no evidence that they contact residual cone processes in the external plexiform layer. The rd mouse is a logical model to study the effects of transplantation of photoreceptors because second- and third-order retinal neurons as well as degenerating cones survive in the adult retina.

Survival and integration of neural retinal transplants in rd mice.

To examine the ultrastructure of the rd retina after transplantation of small micro-aggregates of neural retina in order to determine their survival and integration with the host retina and for sites of communication between transplant and host neurons. Neonatal micro-aggregates from transgenic mice expressing a LacZ gene reporter gene in their rods were transplanted into the subretinal space of transgenic rd mice expressing a LacZ reporter gene in their rod bipolar cells. The mice were killed at various times after transplantation surgery and studied by light and electron microscopy. Retinal transplants survived well, as long as 8 months, without signs of rejection and were well integrated into the host retina. Cell bodies of transplanted rods made membrane-to-membrane contacts with rod bipolar cells of the host at areas where there were gaps in the host external plexiform layer. One synaptic process of a transplanted rod was found on the vitreal side of the host's external limiting membrane. In two cases, a postsynaptic process in a transplanted rod spherule contained an Xgal label, implying that it belonged to a host rod bipolar. There was evidence of extension of processes between host and transplant retinas involving astrocytic rather than neural structures. Retinal allografts to the subretinal space of rd mice survive indefinitely. Close but non-synaptic contacts occur between transplant and host neurons that could allow ephaptic communication between these two retinas. Evidence of synaptic contacts between transplant and host was difficult to find.

Retinal transplantation-induced recovery of retinotectal visual function in a rodent model of retinitis pigmentosa.

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. 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. 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. 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.

Photoreceptor transplantation in retinitis pigmentosa: short-term follow-up

Purpose To explore the use of adult human photoreceptor transplantation as a treatment for advanced retinitis pigmentosa (RP). Design Prospective noncomparative case series. Participants Eight patients with advanced RP. Intervention Transplantation of adult human cadaver photoreceptor sheets harvested with the excimer laser. No immunosuppression was used postoperatively. Patients were followed for 12 months postoperatively. Main outcome measure Visual acuity and retinal function measured by psychophysical, electrophysiologic, and clinical testing. Results Best-corrected visual acuity (Bailey-Lovie chart), median reading speed, contrast sensitivity, and visual fields for the operated eye were not statistically significantly improved postoperatively. The amplitude and latency of the maculoscope electroretinogram, as well as the log threshold for dark adaptation, did not change between the operated and control (unoperated) eye. There was no detectable homograft reaction on slit-lamp biomicroscopy or fluorescein angiography. The only adverse effect observed was one patient who complained of monocular diplopia after retinal transplantation and subsequent cataract surgery. Conclusions Allogeneic adult human photoreceptor transplantation is feasible in RP but was not associated with rescue of central vision or a delay in visual loss. However, any possible slowing in the rate of retinal degeneration will take many years to determine.

細胞治療とＤＤＳ 網膜組織再生治療へのアプローチ

Retinal degenerative disease such as retinitis pigmentosa and age-related macular degeneration causes visual defects with the progress in their diseases. In case of retinitis pigmentosa a large number of mutations in genes that involved in phototransduction pathway have been identified. However pharmacological treatment is presently unavailable for these diseases. Several strategies such as retinal pigment epithelial cell transplantation, growth factor application, vitamin supplementation or gene therapy are under examination for preventing the photoreceptor cell death. Among these strategies, some growth factors are commonly effective on preventing photoreceptor cell death even the mutated genes cause that photoreceptor cell death is different. Is it possible to prevent photoreceptor cell death and to maintain the visual function by continuously supplying growth factors to photoreceptor cells? Which methodology is better to supply growth factor continuously, gene transfer to retina or transplantation? We describe here the approach to prevent photoreceptor cell death by using gene transfected iris pigment epithelial cell transplantation.

Microcontact printing on human tissue for retinal cell transplantation.

To demonstrate that microcontact printing, a modern materials fabrication technique, can be used to engineer the surface of human tissue and to show that inhibitory molecules can be used to pattern the growth of retinal pigment epithelial cells or iris pigment epithelial cells on human lens capsule for transplantation. Photolithographic techniques were used to fabricate photoresist-coated silicon substrates into molds. Poly(dimethylsiloxane)stamps for microcontact printing were made from these molds. The poly(dimethylsiloxane) stamps were then used to "wet-transfer" growth inhibitory molecules to the surface of prepared human lens capsules that were obtained during cataract surgery. Human retinal pigment epithelial and rabbit iris pigment epithelial cells were grown on a lens capsule substrate in the presence and absence of a patterned array of inhibitory factors. We found that human lens capsule could be microprinted with a precision similar to that obtained on glass or synthetic polymers. Retinal pigment epithelial cells and iris pigment epithelial cells cultured onto an untreated lens capsule showed spreading and formed into fusiform-appearing cells. In contrast, cells cultured on a lens capsule with a hexagonal micropattern of growth inhibitory molecules retained an epithelioid form within the inhibitory hexagons. Inhibitory growth molecules can be micropatterned onto human lens capsule, and these micropatterns can control the organization of retinal pigment epithelial cells or iris pigment epithelial cells cultured onto the lens capsule surface. Microprinting on autologous human tissue may facilitate efforts to effectively organize cell cultures and transplantations for the replacement of vital ocular tissues such as the retinal pigment epithelium in age-related macular degeneration.

Preservation of visual responsiveness in the superior colliculus of RCS rats after retinal pigment epithelium cell transplantation

The dystrophic RCS rat undergoes progressive photoreceptor degeneration due to a primary defect in retinal pigment epithelial (RPE) cells. This has a major impact on central visual responsiveness. Here we have examined how functional deterioration is contained by subretinal transplantation of immortalized human RPE cells. Transplantation was done at three to four weeks of age prior to significant photoreceptor loss and recipients were kept on cyclosporin. At six months of age, sensitivity maps and multi-unit response properties were obtained across the visual field by recording at 76 equidistant sites encompassing the whole superior colliculus. A significant degree of functional protection, both in terms of area of responsive retina and response characteristics was observed following RPE transplantation. At best, the sensitivity, latency of onset, and response rise time were all maintained within normal ranges and this was achieved with no more than half of the normal complement of photoreceptors. Although partial, the degree of anatomical preservation (both in terms of outer nuclear layer thickness and area of rescue) correlated well with the level of preserved visual sensitivities. Sham injections also resulted in rescue, though the area of preservation was strictly confined to the needle injury site and the response properties were significantly worse than with RPE injections. This study shows that central physiological responsiveness and correlated retinal morphology can be preserved in an animal model of retinal disease by implantation of an immortalized cell line. The use of retinal sensitivity measurements provides a background for assessing higher visual functions in these animals and a direct comparison for human perimetry measures.

Morphometric analysis of the macula in eyes with disciform age-related macular degeneration.

To evaluate the extent of neural cell death in eyes with disciform age-related macular degeneration. Six eyes with disciform degeneration at various stages and five age-matched control eyes were selected for morphometric analysis using digitized light microscopic images. Disciform scars were classified as subneurosensory retinal, subretinal pigment epithelial, or combined lesions. The nuclei of the ganglion cell, inner nuclear, and outer nuclear layers were counted in contiguous 100 microm segments spanning a distance from 1,500 microm nasal to 1,500 microm temporal to the fovea. The outer nuclear layer was most severely attenuated in eyes with disciform scars, demonstrating a 69.4% reduction in cell number relative to control eyes. A loss in retinal ganglion cells (by 7.3%) and an increase in inner nuclear layer cells (by 10%) were observed, but these changes were not significant. Photoreceptor loss was most pronounced when the disciform scar was not covered by the retinal pigment epithelium. The nuclei of the outer nuclear layer are significantly attenuated in eyes with disciform age-related macular degeneration, while the ganglion cell and inner nuclear layers are relatively preserved. These findings suggest that replacement of outer nuclear function, by either retinal transplantation or implantation of the intraocular retinal prosthesis, might be a feasible therapeutic option for patients with this condition.

Morphometric analysis of the macula in eyes with geographic atrophy due to age-related macular degeneration.

To evaluate the extent of neural cell death in eyes with geographic atrophy (GA). Ten eyes with GA and five age-matched control eyes were selected for morphometric analysis. The nuclei of the ganglion cell, inner nuclear, and outer nuclear layers were counted in contiguous 100-microm segments from 1,500 microm nasal to 1,500 microm temporal to the fovea. The outer nuclear layer was most severely attenuated in eyes with GA, demonstrating a 76.9% reduction relative to control eyes (P < 0.0001). A significant loss of ganglion cells (by 30.7%) was also observed (P = 0.0008). There was no significant difference in the inner nuclear layer cells (P = 0.30). Among the GA eyes, the nuclei in all three layers were significantly reduced in segments in which the retinal pigment epithelium was completely absent (P </= 0.0003). Although the nuclei of the outer nuclear layer in eyes with GA were markedly attenuated, the nuclei of the inner nuclear layer were relatively preserved. There was also a significant reduction in ganglion cells in GA eyes, but considerable numbers remained even in the areas of complete retinal pigment epithelium atrophy. This finding suggests that therapies aimed at replacing outer nuclear function (such as neural retinal and retinal pigment epithelium transplantation or implantation of the intraocular retinal prosthesis) may be feasible for restoring vision in these patients.

Evaluation of viability of retinal photoreceptor cells by using their endogenous electrical field

The rod photoreceptor cells are electrical dipoles sustained by cell metabolic energy. Polarity of photoreceptor cells is directly connected to the so-called “dark current” which circulate along the living photoreceptors. Since only the living cells in a good functional state display electrical polarity, the orientation of photoreceptors in static electric field reflects their viability as long as it depends on the functionality of molecular mechanisms that maintain the dark current. Studying the rod cells' orientation in static electric field at different times after their isolation is thus an accurate way to evaluate the cell viability/degeneration. Retinal transplant experiments in animals and humans, which are presently in progress, require a quick and reliable viability test of cells/tissue to be transplanted. Checking the orientation pattern of rod photoreceptors in static electric field prior to transplantation is a candidate method for an accurate cell viability test.

Absorbable sandwich-like membrane for retinal-sheet transplantation.

Neural retinal transplantation has great potential for the alleviation of different degenerative and hereditary retinal disorders. However, because of the fragile and soft nature of retina, retinal-sheet transplantation is relatively difficult to achieve. To overcome this difficulty, we developed a technique for lamellar tissue transplantation. Biodegradable gelatin membranes were fabricated into a sandwich and encapsulated retinal grafts for transplantation. Before transplantation, we characterized the in vivo and in vitro properties of such membranes to determine the optimal sterilization procedure, that is, a sterile membrane with suitable degradability and good mechanical properties and without cytotoxicity. Three sterilization methods were conducted, with hydrogen peroxide gas plasma (HPGP), ethylene oxide (EO), and gamma-ray irradiation (gamma). The results were compared with those of a control (no disinfection). Initial studies revealed that the gelatin membranes sterilized with HPGP or EO exhibited retinal pigment epithelium (RPE) cytotoxicity, whereas the membrane sterilized by 16.6-kGy gamma ray irradiation had no RPE cytotoxicity and had enhanced mechanical properties. In the in vivo rabbit study, implanted gelatin membranes demonstrated satisfactory biocompatibility without any inflammation. Transplanted retinal sheets survived well and developed laminar structures. Such a method using gelatin membranes for tissue transportation has great potential for future routine retinal-sheet transplantation.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue. Haruta M, Kosaka M, Kanegae Y, Saito I, Inoue T, Kageyama R, Nishida A, Honda Y, Takahashi M. Nat Neurosci 2001; DOI:10.1038/nn762.

During eye development, the inner layer of the optic cup differentiates into the neural retina and iris pigment epithelium. When grown in tissue culture, rat iris derived cells express neurofilament when in an environment that promotes retinal cell differentiation. These cell express rhodopsin if infected with a Crx-transducing adenovirus. These transfected cells are also morphologically similar to photoreceptors. In contrast, Crx-transduced rat hippocampus stem cells are not able to express rhodopsin. Crx-transduced ciliary margin cells express rhodopsin, similar to iris-derived cells. This ability might be related to the embryonic origin of the iris and ciliary body cells. These iris and ciliary cell lines may potentially be used in retinal transplantation.—Hans E. Grossniklaus

Transplantation of allogenic anterior lens capsule to the subretinal space in pigs.

To investigate the consequences of transplantation of a new basement membrane to the subretinal space (SRS) as a substitution of Bruch's membrane. Porcine anterior lens capsules (ALC) were transplanted to the subretinal space of 20 eyes from 19 young Danish landrace pigs. All pigs underwent a three port localized pars plana vitrectomy. Seventeen eyes received naked ALC. In three experiments the ALC was embedded in gelatine, in order to prevent curling of the ALC. The observation period varied between zero and 49 days. The pigs were examined by ophthalmoscopy and fundus photography. Histopathological examination of enucleated eyes was performed at the end of the experiment. ALCs transplanted to the subretinal space were well-tolerated and caused no inflammation when Bruch's membrane was left undamaged. After 11 days host RPE and glial cells started to cover the ALC in a competitive fashion. When Bruch's membrane was damaged, ingrowths of choroidal vessels and fibroblasts was prominent. The use of gelatine to flatten the ALC did not prevent curling, and gelatine caused pronounced inflammation. It is possible to transplant porcine ALC to the SRS of the pig. ALCs are well-tolerated in the SRS and are covered with well-differentiated monolayers of host RPE-cells, if Bruch's membrane is left intact.

Immunobiology and privilege of neuronal retina and pigment epithelium transplants

Despite the existence of ocular immune privilege, immune rejection may be a barrier to successful retinal transplantation. We have examined in mice the extent to which the subretinal space (SRS) is an immune privileged site, and whether retinal pigment epithelium and neuronal retinal tissue have properties of immune privileged tissues. We report that (1) The SRS is an immune privileged site; (2) Neonatal RPE is an immune privileged tissue; (3) Neuronal retina is a partially immune privileged tissue; and (4) Microglia within neonatal neural retina grafts promote photoreceptor differentiation, become activated, and induce sensitization of the recipient and serve as targets of immune rejection.

Retinal transplantation--advantages of intact fetal sheets.

Retinal transplantation aims to prevent blindness and to restore eyesight, i.e., to rescue photoreceptors or to replace damaged photoreceptors with the hope of reestablishing neural circuitry. Retinal donor tissue has been transplanted as dissociated cells or intact sheets. A promising experimental paradigm is the subretinal transplantation of sheets of fetal retina with or without its attached retinal pigment epithelium (RPE) into recipient rats with retinal degeneration. As long as healthy RPE either from the host or from the graft is present, such transplants can develop lamination resembling a normal retina. Different methods have been used to demonstrate transplant/host connectivity. In two different rat retinal degeneration models, visually evoked responses can be demonstrated in an area of the superior colliculus corresponding to the placement of the transplant in the retina. In summary, sheets of fetal retina can morphologically repair an area of a degenerated retina, and there is evidence to suggest that transplants form synaptic connections with the host and restore visual responses in blind rats.

The polarity of the retinal pigment epithelium.

The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.

Müller cells in long-term full-thickness retinal transplants.

Müller cells are essential in creating and maintaining intricate neuroretinal architecture. The functions of this important glial cell are not limited to mere support of the retinal neurons, but also include interaction in synaptic transmission and activation in response to retinal insult. In this study, we have examined Müller cell morphology and degree of activation in embryonic full-thickness rabbit neuroretinal grafts, which were positioned under the host retina using vitrectomy technique. After surviving 3-10 months, retinal specimens were examined with hematoxylin and eosin staining and immunohistochemical analysis of vimentin and glial fibrillary acidic protein (GFAP) expression. In the host retina covering the graft, outer layers were degenerated, and vimentin-labeled Müller cells in this area appeared short, disorganized, and displayed strong GFAP labeling. In the graft, vimentin-labeled Müller cells spanning the retinal layers in the normal manner were found. Müller cells in 3-month grafts were well labeled by GFAP, whereas in older grafts, GFAP labeling was very weak or absent. Our results suggest that Müller cells in well-laminated full-thickness retinal grafts display many of the normal morphological features and retain a normal organization even after prolonged survival times. The loss of the initial degree of Müller cell activation indicates a long-term stability of the graft. The degeneration and gliosis of the host retina covering the graft is best explained by the merangiotic nature of the rabbit retina and may limit the usefulness of the rabbit in retinal transplantation experiments.

The proliferative and apoptotic activities of E2F1 in the mouse retina.

The E2F1 transcription factor controls cell proliferation and apoptosis. E2F1 activity is negatively regulated by the retinoblastoma (RB) protein. To study how inactivation of Rb and dysregulated E2F1 affects the developing retina, we analysed wild-type and Rb(-/-) embryonic retinas and retinal transplants and we established transgenic mice expressing human E2F1 in retinal photoreceptor cells under the regulation of the IRBP promoter (TgIRBPE2F1). A marked increase in cell proliferation and apoptosis was observed in the retinas of Rb(-/-) mice and TgIRBPE2F1 transgenic mice. In the transgenic mice, photoreceptor cells formed rosette-like arrangements at postnatal days 9 through 28. Complete loss of photoreceptors followed in the TgIRBPE2F1 mice but not in the Rb(-/-) retinal transplants. Both RB-deficient and E2F1-overexpressing photoreceptor cells expressed rhodopsin, a marker of terminal differentiation. Loss of p53 partially reduced the apoptosis and resulted in transient hyperplasia of multiple cell types in the TgIRBPE2F1 retinas at postnatal day 6. Our findings support the concept that cross-talk occurs between different retinal cell types and that multiple genetic pathways must become dysregulated for the full oncogenic transformation of neuronal retinal cells.

Antigenicity and immunogenicity of allogeneic retinal transplants.

The transplantation of neuronal cells and tissues represents a promising approach for the treatment of incurable neurodegenerative diseases. Indeed, it has been reported recently that retinal transplantation can rescue photoreceptor cells and delay age-related changes in various retinal layers in rodents. However, retinal grafts deteriorate progressively after placement in recipients' eyes. Here we investigated whether a host's immune response elicited toward the graft contributes to its deterioration. Using an ELISA spot assay, we measured T cell responses to retinal tissues placed in the vitreous cavity of syngeneic and allogeneic mice. We found that allogeneic retinas induced potent alloimmune responses mediated by T cells secreting type 1 cytokines (IFN-gamma and IL-2). No response was found in mice engrafted with syngeneic retinas. In addition, all syngeneic retinal grafts displayed no signs of tissue damage (at 55 days), while the majority of allogeneic retinas deteriorated as early as 12 days after placement. Next, we showed that anti-donor responses occurred within two phenotypically and functionally distinct T cell subsets: CD4+ T cells secreting IL-2 and CD8+ T cells producing IFN-gamma. Importantly, CD4+ T cells were necessary and sufficient to cause graft deterioration, while CD8+ T cells did not contribute to this process.