Retinal morphological and functional changes in an animal model of retinitis pigmentosa.

The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.

Orientation-specific modulation of rat retinal ganglion cell responses and its dependence on relative orientations of the center and surround gratings.

In the primary visual cortex (V1), it has been shown that the neuronal response elicited by a grating patch in the receptive field (RF) center can be suppressed or facilitated by an annular grating presented in the RF surround area; the effect depends on the relative orientations of the two gratings. The effect is thought to play a role in figure-ground segregation. Here we have found that response modulation similar to that reported in cortical area V1 can also be found in all major classes of retinal ganglion cells (RGCs), including "concentric" cells. Orientation-specific response modulation of this kind cannot result from interactions of independent RF mechanisms; therefore more complex mechanism, which takes into account the relative orientations of the gratings in the RF center and surround, or sensing the borders between texture regions, has to be present in RFs of RGCs, even of the concentric type. This challenges the consensus notion that their responses to visual stimuli are governed entirely by a RF composed of separate mechanisms: center, antagonistic surround, and modulatory extraclassical surround. Our findings raise the question of whether initial stages of complex analysis of visual input, normally attributed to the visual cortex, can be achieved within the retina.

Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration.

Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early-onset blindness. Here we show long-term functional rescue using hESC-derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice-compliant hESC-RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose-dependent fashion without teratoma formation or untoward pathological reactions. Near-normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice-compliant study was carried out in the NIH III immune-deficient mouse model. Long-term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/tumor formation after subretinal hESC-RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases.

Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration.

PURPOSE: It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. METHODS: Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. RESULTS: Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. CONCLUSIONS: RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Syngeneic Schwann cell transplantation preserves vision in RCS rat without immunosuppression.

PURPOSE: To evaluate the efficacy of immunologically compatible Schwann cells transplanted without immunosuppression in the RCS rat retina to preserve vision. METHODS: Syngeneic (dystrophic RCS) Schwann cells harvested from sciatic nerves were cultured and transplanted into one eye of dystrophic RCS rats at an early stage of retinal degeneration. Allogeneic (Long-Evans) Schwann cells and unoperated eyes served as controls. Vision through transplanted and unoperated eyes was then quantified using two visual behavior tasks, one measuring the spatial frequency and contrast sensitivity thresholds of the optokinetic response (OKR) and the other measuring grating acuity in a perception task. RESULTS: Spatial frequency thresholds measured through syngeneically transplanted eyes maintained near normal spatial frequency sensitivity for approximately 30 weeks, whereas thresholds through control eyes deteriorated to less than 20% of normal over the same period. Contrast sensitivity was preserved through syngeneically transplanted eyes better than through allogeneic and unoperated eyes, at all spatial frequencies. Grating acuity measured through syngeneically transplanted eyes was maintained at approximately 60% of normal, whereas acuity of allogeneically transplanted eyes was significantly lower at approximately 40% of normal. CONCLUSIONS: The ability of immunoprivileged Schwann cell transplants to preserve vision in RCS rats indicates that transplantation of syngeneic Schwann cells holds promise as a preventive treatment for retinal degenerative disease.

Intraocular CNTF reduces vision in normal rats in a dose-dependent manner.

PURPOSE: CNTF is a neuroprotective agent for retinal degenerations that can cause reduced electroretinogram (ERG) amplitudes. The goal of the present study was to determine the effects of intraocular delivery of CNTF on normal rat visual function. METHODS: Full-field scotopic and photopic ERG amplitudes and spatial frequency thresholds of the optokinetic response (OKR) of adult Long-Evans rats were measured before and after intravitreous injection of CNTF or subretinal delivery of adenoassociated virus-vectored CNTF (AAV-CNTF) into one eye. Visual acuity was also measured by using the Visual Water Task in AAV-CNTF-injected animals. Multiunit luminance thresholds were recorded in the superior colliculus after CNTF injection, and the eyes were examined histologically. RESULTS: In eyes injected with a high dose of CNTF, ERG amplitudes and OKR thresholds measured through CNTF-injected eyes were decreased by 45% to 70% within 6 days after injection. ERG amplitudes had begun to recover by 21 days, whereas OKR thresholds only began to recover after 56 days. Neither OKR thresholds nor ERG amplitudes fully recovered until 90 to 100 days. When measured in the superior colliculus at 2 weeks after CNTF injection, luminance thresholds were elevated by 0.35 log units. In AAV-CNTF-injected eyes, OKR thresholds, and visual acuity were reduced by approximately 50% for at least 6 months, and scotopic and photopic ERG b-waves were reduced by 30% to 50%. Photoreceptor loss occurred in the injected regions in some of the eyes. By contrast, comparison of dose-response analysis with a dose-response study of light damage strongly suggests that therapeutic doses of CNTF exist that do not suppress ERG responses. CONCLUSIONS: Intraocular delivery of CNTF, which preserves photoreceptors in animal models of retinal degeneration, impairs visual function in normal rats at very high doses, but not at lower doses that still provide protection from constant light damage.

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

Transplanted olfactory ensheathing cells promote regeneration of cut adult rat optic nerve axons.

Transplantation of olfactory ensheathing cells into spinal cord lesions promotes regeneration of cut axons into terminal fields and functional recovery. This repair involves the formation of a peripheral nerve-like bridge in which perineurial-like fibroblasts are organized into a longitudinal stack of parallel tubular channels, some of which contain regenerating axons enwrapped by Schwann-like olfactory ensheathing cells. The present study examines whether cut retinal ganglion cell axons will also respond to these cells, and if so, whether they form the same type of arrangement. In adult rats, the optic nerve was completely severed behind the optic disc, and a matrix containing cultured olfactory ensheathing cells was inserted between the proximal and distal stumps. After 6 months, the transplanted cells had migrated for up to 10 mm into the distal stump. Anterograde labeling with cholera toxin B showed that cut retinal ganglion cell axons had regenerated through the transplants, entered the distal stump, and elongated for 10 mm together with the transplanted cells. Electron microscopy showed that a peripheral nerve-like tissue had been formed, similar to that seen in the spinal cord transplants. However, in contrast to the spinal cord, the axons did not reach the terminal fields, but terminated in large vesicle-filled expansions beyond which the distal optic nerve stump was reduced to a densely interwoven mass of astrocytic processes.

Morphological changes in the Royal College of Surgeons rat retina during photoreceptor degeneration and after cell-based therapy.

There are concomitant morphological and functional changes in the inner retina during the course of photoreceptor degeneration in a range of animal models of retina degeneration and in humans with eye disease. One concern that has been raised is that the changes occurring in the inner retina might compromise attempts to rescue or restore visual input by various interventional approaches. It is known that cell-based therapy can preserve significant visual capability for many months. In this study, we examine the overall changes in the Royal College of Surgeons (RCS) rat during degeneration and the effects of cell transplantation by means of immunohistochemistry and confocal microscopy. The degenerative changes are complex, and they progress with age. They involve the neurons with which both rods and cones interconnect--retinal second- and third-order neurons underwent dramatic modification, including sprouting, retraction as photoreceptor loss progressed--as well as Müller glia and secondary vascular changes, which were associated at later times with neuronal migration. The pathological vascular changes led to major disruption of inner retina. After introducing a retinal pigment epithelial cell line to the subretinal space early in the progress of photoreceptor degeneration, most inner retinal changes were held in abeyance for up to at least 10 months of age. Given the concern that has been raised regarding whether inner retinal changes might compromise any graft-related benefit, this is an encouraging finding.

Grafting of ARPE-19 and Schwann cells to the subretinal space in RCS rats.

PURPOSE: To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. METHODS: Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. RESULTS: Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. CONCLUSIONS: Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

Abnormal c-fos-like immunoreactivity in the superior colliculus and other subcortical visual centers of pigmented Royal College of Surgeons rats.

Neurons in the central nervous system often show a transient up-regulation of expression of the immediate early gene c-fos when presented with precise novel stimuli. In normal rats, neurons in most subcortical visual centers show low levels of fos-like immunoreactivity (FLI) expression, but there is a substantial and transient increase in FLI expression if the animal is exposed to a flashing light. This is especially evident in the superior colliculus (SC). We have examined here FLI expression in the subcortical visual centers of the Royal College of Surgeons rat, focusing specifically on the SC. In this animal, as a result of a genetic defect, there is early loss of rod photoreceptors over the first few months of life, along with slower disappearance of cones. Although light stimulation showed that FLI expression was very similar to that seen in normal rats, the basal levels of FLI expression under dark-maintained conditions were much higher than normal, even exceeding the levels seen after visual stimulation. In the SC, the elevation of FLI expression was already evident by 6 weeks of age and reached a plateau by 17 weeks. Other subcortical visual centers also showed elevated basal levels of FLI expression, although in general the increases were less dramatic than the increase in the SC. The elevated FLI expression in dark-maintained condition seen in the SC was abolished by contralateral optic nerve section. It was also severely diminished by subretinal cell transplantation at 3 weeks of age with the objective of limiting photoreceptor loss over part of the retina. These results suggest that the elevated basal FLI expression is a retina-driven event. Although it correlates with the loss of rod photoreceptors, it is unlikely to reflect reduced photoreceptor drive but rather some form of bursting activity generated in the inner retina, as a result of circuit reorganization or receptor up-regulation.

Quantification of spatial vision in the Royal College of Surgeons rat.

PURPOSE: To examine how spatial vision deteriorates in the RCS rat over time as a background to experimental studies aimed at limiting photoreceptor degeneration. METHODS: The Visual Water Task was used to quantify the grating acuity of pigmented dystrophic RCS rats as they aged and to compare both grating acuity and contrast sensitivity in nondystrophic RCS rats with those parameters in normal pigmented laboratory rats (Long-Evans). RESULTS: Nondystrophic rats had grating acuities and contrast sensitivity functions that were similar to those obtained from Long-Evans rats. The grating acuity of dystrophic rats deteriorated from 80% of normal at 1 month of age to blindness by 11 months. Acuity declined rapidly to 0.32 cyc/deg over the first 4 months, with a slower decline thereafter. CONCLUSIONS: Robust measures of vision can be achieved in RCS rats using the Visual Water Task, and with this test, no visual dysfunction can be detected in the background strain. The course of functional deterioration in dystrophic rats is highly predictable, allowing the approach to be used to explore the substrates of the deterioration in vision and to monitor the effects of therapeutic retinal interventions on spatial vision.

Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies.

PURPOSE: To develop, by specific genetic modification, a differentiated human retinal pigment epithelial (RPE) cell line with an extended life span that can be used for investigating their function in vitro and for in vivo transplantation studies. METHODS: Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-19. Factors reported to be important in RPE and photoreceptor cell function and survival in vivo were examined. RESULTS: Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology, expressed cytokeratins, and displayed junctional distribution of ZO-1, p100-p120 and beta-catenin. The cells expressed mRNA for RPE65 and cellular retinaldehyde-binding protein (CRALBP) and the trophic and growth factors brain-derived neurotropic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-alpha, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF, bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59. After activation with IFN-gamma both HLA-ABC and CD54 were upregulated, and the expression of HLA-DR was induced. Both cell lines failed to express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte reaction were unable to induce T-cell proliferation. Fas ligand (CD95L) was not detected in vitro by RT-PCR. Similar results were obtained with the ARPE-19 cell line. CONCLUSIONS: RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.

Reinnervation of the pretectum in adult rats by regenerated retinal ganglion cell axons: anatomical and functional studies.

We have investigated the specificity of reinnervation and terminal arborization of injured retinal ganglion cell (RGC) axons in the brainstem with the object of studying in a simple situation the degree to which regenerating axons are able to replicate the characteristic patterns of terminal arborization and restore normal function. We have focussed here on the pathway that is responsible for the pupillary light reflex, which is mediated through the olivary pretectal nucleus (OPN). In adult rats, the left optic nerve was transected and a segment of peripheral nerve (PN) graft was used to bridge between the retina and different regions of the ipsilateral brainstem, including the superior colliculus. After 4-13 months, regenerated RGC axons were examined in coronal sections stained for cholera toxin B subunit. RGC axons were found extending into the ipsilateral brainstem for distances of up to 6 mm. Within the pretectum, axons innervated the OPN and the nucleus of the optic tract preferentially, and formed distinctive terminal arbors within each. Within the SC axons extended laterally into the visual layers and formed a different type of arborization. On testing the pupillary light reflex, it was found in best cases to show response amplitudes which were comparable to those recorded from control intact animals. However, unlike normals, the response amplitude tended to diminish with repeated stimulation and also appeared to deteriorate with age, although responses could still be detected in some cases as long as 15 months after grafting. These results indicate that regenerating axons can selectively reinnervate denervated nuclei, where they form typical terminal arborizations, and provide the substrates for restoring functional circuitry.

Increased c-fos-like immunoreactivity in the superior colliculus and lateral geniculate nucleus of the rd mouse.

In most subcortical visual centers in normal mice maintained for a period in the dark, very few neurons express fos-like immunoreactivity (FLI), most likely reflecting c-fos expression, but if an animal is exposed to a flashing light, there is transient increase in the number of FLI-expressing cells. In dark-maintained retinal degeneration (rd) mice, with an inherited photoreceptor defect, numbers of FLI-positive cells, identified immunohistochemically, are anomalously elevated in the superior colliculus (SC) and lateral geniculate nucleus (LGN). Eye removal largely prevents the elevated counts. The difference in number of FLI-positive cells in the SC of rd mice and nondystrophic controls is highly significant (p<0.001). Because we have previously found a similar phenomenon in Royal College of Surgeons (RCS) rats, in which photoreceptor loss is caused by a retinal pigment cell defect, it argues for an effect related to photoreceptor loss rather than its cause.

Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

PURPOSE: Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. METHODS: The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). RESULTS: The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. CONCLUSIONS: This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Cell transplantation to arrest early changes in an ush2a animal model.

Purpose. Usher's syndrome is a combined deafness and blindness disorder caused by mutations in several genes with functions in both the retina and the ear. Here the authors studied morphologic and functional changes in an animal model, the Ush2a mouse, and explored whether transplantation of forebrain-derived progenitor cells might affect the progress of morphologic and functional deterioration. Methods. Ush2a mice were tested at postnatal days (P) 70 to P727 using an optomotor test, which provides a repeatable method of estimating rodent visual acuity and contrast sensitivity. A group of mice that received grafts of forebrain-derived progenitor cells at P80 was tested for up to 10 weeks after grafting. At the end of testing, animals were killed, and eyes were processed for histology. Results. The optomotor test showed that both acuity and contrast sensitivity deteriorated over time; contrast sensitivity showed a deficit even at P70. By contrast, photoreceptor loss was only evident later than 1 year of age, though changes in the intracellular distribution of red/green cone opsin were observed as early as P80. Mice that received transplanted cells performed significantly better than control mice and no longer demonstrated abnormal distribution of red/green opsin where the donor cells were distributed. Conclusions. This study showed that vision impairment was detected well before significant photoreceptor loss and was correlated with abnormal distribution of a cone pigment. Cell transplantation prevented functional deterioration for at least 10 weeks and reversed the mislocalization of cone pigment.

Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology.

BACKGROUND: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.

Subretinal transplantation of forebrain progenitor cells in nonhuman primates: survival and intact retinal function.

PURPOSE: Cell-based therapy rescues retinal structure and function in rodent models of retinal disease, but translation to clinical practice will require more information about the consequences of transplantation in an eye closely resembling the human eye. The authors explored donor cell behavior using human cortical neural progenitor cells (hNPC(ctx)) introduced into the subretinal space of normal rhesus macaques. METHODS: hNPC(ctx) transduced with green fluorescent protein (hNPC(ctx)-GFP) were delivered bilaterally into the subretinal space of six normal adult rhesus macaques under conditions paralleling those of the human operating room. Outcome measures included clinical parameters of surgical success, multifocal electroretinogram (mfERG), and histopathologic analyses performed between 3 and 39 days after engraftment. To test the effects of GFP transduction on cell bioactivity, hNPC(ctx)-GFP from the same batch were also injected into Royal College of Surgeons (RCS) rats and compared with nonlabeled hNPC(ctx). RESULTS: Studies using RCS rats indicated that GFP transduction did not alter the ability of the cells to rescue vision. After cells were introduced into the monkey subretinal space by a pars plana transvitreal approach, the resultant detachment was rapidly resolved, and retinal function showed little or no disturbance in mfERG recordings. Retinal structure was unaffected and no signs of inflammation or rejection were seen. Donor cells survived as a single layer in the subretinal space, and no cells migrated into the inner retina. CONCLUSIONS: Human neural progenitor cells can be introduced into a primate eye without complication using an approach that would be suitable for extrapolation to human patients.

Long-term vision rescue by human neural progenitors in a rat model of photoreceptor degeneration.

PURPOSE: As a follow-up to previous studies showing that human cortical neural progenitor cells (hNPC(ctx)) can sustain vision for at least 70 days after injection into the subretinal space of Royal College of Surgeons (RCS) rats, the authors examined how functional rescue is preserved over long periods and how this relates to retinal integrity and donor cell survival. METHODS: Pigmented dystrophic RCS rats (n = 15) received unilateral subretinal injections of hNPC(ctx) at postnatal day (P) 21; control rats (n = 10) received medium alone and were untreated. All animals were maintained on oral cyclosporine A. Function was monitored serially by measuring acuity (using an optomotor test) and luminance thresholds (recording from the superior colliculus) at approximately P90, P150, and P280. Eyes were processed for histologic study after functional tests. RESULTS: Acuity and luminance thresholds were significantly better in hNPC(ctx)-treated animals than in controls (P < 0.001) at all time points studied. Acuity was greater than 90%, 82%, and 37% of normal at P90, P150, and P270, whereas luminance thresholds in the area of best rescue remained similar the whole time. Histologic studies revealed substantial photoreceptor rescue, even up to P280, despite progressive deterioration in rod and cone morphology. Donor cells were still present at P280, and no sign of donor cell overgrowth was seen. CONCLUSIONS: Long-term rescue of function and associated morphologic substrates was seen, together with donor cell survival even in the xenograft paradigm. This is encouraging when exploring further the potential for the application of hNPC(ctx) in treating retinal disease.