Immune Responses to Sequential Binocular Transplantation of Allogeneic Retinal Progenitor Cells to the Vitreous Cavity in Mice.

Intravitreal transplantation of allogeneic human retinal progenitor cells (hRPCs) holds promise as a treatment for blinding retinal degenerations. Prior work has shown that neural progenitors are well-tolerated as allografts following single injections; however, sequential delivery of allogeneic cells raises the potential risk of host sensitization with subsequent immune rejection of grafts. The current study was designed to assess whether an immune response would be induced by repeated intravitreal transplants of allogeneic RPCs utilizing the mouse animal model. We injected murine retinal progenitor cells (gmRPCs), originally derived from donors with a C57BL/6 genetic background, into BALB/c recipient mice in order to provide safety data as to what might be expected following repeated treatment of patients with allogeneic human cell product. Immune responses to gmRPCs were mild, consisting of T cells, B cells, neutrophils, and natural killer cells, with macrophages clearly the predominating. Animals treated with repeat doses of gmRPCs did not show evidence of sensitization, nor was there immune-mediated destruction of the grafts. Despite the absence of immunosuppressive treatments, allogeneic gmRPC grafts survived following repeat dosing, thus providing support for the preliminary observation that repeated injection of allogeneic RPCs to the vitreous cavity is tolerated in patients with retinitis pigmentosa.

Potential therapeutic strategies for photoreceptor degeneration: the path to restore vision.

Photoreceptors (PRs), as the most abundant and light-sensing cells of the neuroretina, are responsible for converting light into electrical signals that can be interpreted by the brain. PR degeneration, including morphological and functional impairment of these cells, causes significant diminution of the retina's ability to detect light, with consequent loss of vision. Recent findings in ocular regenerative medicine have opened promising avenues to apply neuroprotective therapy, gene therapy, cell replacement therapy, and visual prostheses to the challenge of restoring vision. However, successful visual restoration in the clinical setting requires application of these therapeutic approaches at the appropriate stage of the retinal degeneration. In this review, firstly, we discuss the mechanisms of PR degeneration by focusing on the molecular mechanisms underlying cell death. Subsequently, innovations, recent developments, and promising treatments based on the stage of disorder progression are further explored. Then, the challenges to be addressed before implementation of these therapies in clinical practice are considered. Finally, potential solutions to overcome the current limitations of this growing research area are suggested. Overall, the majority of current treatment modalities are still at an early stage of development and require extensive additional studies, both pre-clinical and clinical, before full restoration of visual function in PR degeneration diseases can be realized.

Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells.

PURPOSE: Differentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents. METHODS: Using porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR. RESULTS: The microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4) decreased sharply (>twofold). The specific dynamics of expression changes varied according to the transcript and treatment condition over the five days examined following induction. The magnitude of neuronal marker induction was greater for the ciliary neurotrophic factor condition while glial fibrillary acidic protein induction was greater for the FBS condition. CONCLUSIONS: The transient dynamic of CXCR4 expression during induction of differentiation, as well as the upregulation of several major histocompatibility complex (MHC) transcripts, has implications in terms of graft integration and tolerance, respectively. These data confirm and extend in the pig the findings previously reported with murine retinal progenitors and support the use of this large animal model for translational development of regenerative approaches to neurologic diseases.

Age-Related Macular Degeneration (AMD) Transmitochondrial Cybrids Protected from Cellular Damage and Death by Human Retinal Progenitor Cells (hRPCs).

PURPOSE: One of the leading causes of irreversible blindness worldwide, age-related macular degeneration (AMD) is a progressive disorder leading to retinal degeneration. While several treatment options exist for the exudative form of AMD, there are currently no FDA-approved treatments for the more common nonexudative (atrophic) form. Mounting evidence suggests that mitochondrial damage and retinal pigment epithelium (RPE) cell death are linked to the pathogenesis of AMD. Human retinal progenitor cells (hRPCs) have been studied as a potential restorative therapy for degenerative conditions of the retina; however, the effects of hRPC treatment on retinal cell survival in AMD have not been elucidated. METHODS: In this study, we used a cell coculture system consisting of hRPCs and AMD or age-matched normal cybrid cells to characterize the effects of hRPCs in protecting AMD cybrids from cellular and mitochondrial damage and death. RESULTS: AMD cybrids cocultured with hRPCs showed (1) increased cell viability; (2) decreased gene expression related to apoptosis, autophagy, endoplasmic reticulum (ER) stress, and antioxidant pathways; and (3) downregulation of mitochondrial replication genes compared to AMD cybrids without hRPC treatment. Furthermore, hRPCs cocultured with AMD cybrids showed upregulation of (1) neuronal and glial markers, as well as (2) putative neuroprotective factors, responses not found when hRPCs were cocultured with age-matched normal cybrids. CONCLUSION: The current study provides the first evidence that therapeutic benefits may be obtainable using a progenitor cell-based approach for atrophic AMD. Our results suggest that bidirectional interactions exist between hRPCs and AMD cybrids such that hRPCs release trophic factors that protect the cybrids against the cellular and mitochondrial changes involved in AMD pathogenesis while, conversely, AMD cybrids upregulate the release of these neuroprotective factors by hRPCs while promoting hRPC differentiation. These in vitro data provide evidence that hRPCs may have therapeutic potential in atrophic AMD.

Effects of glial cell line-derived neurotrophic factor on cultured murine retinal progenitor cells.

PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective of retinal neurons, and transduced retinal progenitor cells (RPCs) can deliver this cytokine for the treatment of retinal diseases, yet the potential effects of GDNF on RPCs have received little attention. METHODS: Murine RPCs were assessed under multiple conditions in the presence or absence of epidermal growth factor (EGF, 20 ng/ml) and/or GDNF (10 ng/ml) using a variety of techniques, including live-cell imaging, caspase-3 activity assay, whole genome microarray, quantitative polymerase chain reaction (qPCR), and western blotting. RESULTS: Live monitoring revealed that formation of initial aggregates resulted largely from the collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres enlarged in size and number, with more reaching the threshold criteria for cross-sectional areas in the EGF+GDNF condition. Proliferation was measurably augmented in association with EGF+GDNF, and K(i)-67 expression was modestly increased (1.07 fold), as were hairy and enhancer of split 5 (Hes5), mammalian achaete-scute homolog 1 (Mash1), and Vimentin. However, global gene expression did not reveal a notable treatment-related response, and the expression of the majority of progenitor and lineage markers examined remained stable. GDNF reduced RPC apoptosis, compared to complete growth-factor withdrawal, although it could not by itself sustain mitotic activity. CONCLUSIONS: These data support the feasibility of developing GDNF-transduced RPCs as potential therapeutic agents for use in retinal diseases.

Laser injury promotes migration and integration of retinal progenitor cells into host retina.

PURPOSE: The migration and integration of grafted cells into diseased host tissue remains a critical challenge, particularly in the field of retinal progenitor cell (RPC) transplantation. It seems that natural physical barriers at the outer retina can impede the migration of grafted RPCs into the host retina. The purpose of this study was to investigate the integration and differentiation of murine RPCs transplanted into the subretinal space of mice with laser-induced damage to the outer retina. METHODS: RPCs were harvested from the neural retinas of postnatal day 1 enhanced green fluorescent protein (GFP) mice. Retinal photocoagulation was performed using a diode laser. Two microl containing approximately 6x10(5) expanded RPCs in suspension were injected into the subretinal space of the recipient animals following laser treatment. Cell morphometry was performed to assess the integration of donor cells. Immunohistochemistry and western blot were performed on recipient retinas. RESULTS: Three weeks after transplantation, 1,158+/-320 cells per eye had migrated into the recipient outer nuclear layer (ONL). Most of these cells resided in the ONL around the retinal laser lesion. A subpopulation of these cells developed morphological features reminiscent of mature photoreceptors, expressed photoreceptor specific proteins including synaptic protein, and appeared to form synaptic connections with bipolar neurons. Retinal photocoagulation resulted in a significantly increased expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and cluster differentiation 44 (CD44s), and a decreased expression of neurocan. CONCLUSIONS: Transplanted RPCs migrate and integrate into the laser-injured ONL where they differentiate into photoreceptors with morphological features reminiscent of mature photoreceptors, express synaptic protein, and appear to form synaptic connections with retinal bipolar neurons. Following retinal photocoagulation, the enhanced level of integration of grafted RPCs is partially associated with increased expression of MMP-2 and, to a lesser extent, MMP-9, together with decreased levels of inhibitory molecules.

Enhanced differentiation of retinal progenitor cells using microfabricated topographical cues.

Due to the retina's inability to replace photoreceptors lost during retinal degeneration, significant interest has been placed in methods to implant replacement cells. Polymer scaffolds are increasingly being studied as vehicles for cellular delivery to degenerated retinas. Previously, we fabricated poly(methyl methacrylate) thin film scaffolds that increased survival and integration of implanted retinal progenitor cells (RPCs). Additionally, these scaffolds minimized the trauma and cellular response associated with implantation of foreign bodies into mouse eyes. Here, we demonstrate that biodegradable polycaprolactone (PCL) thin film scaffolds can be fabricated with integrated microtopography. Microfabricated topography in a PCL thin film enhanced the attachment and organization of RPCs compared to unstructured surfaces. Using real-time quantitative polymerase chain reaction we also observed that attachment to microtopography induced cellular differentiation. RPCs grown on PCL thin films exhibited an increase in gene expression for the photoreceptor markers recoverin and rhodopsin, an increase in the glial and Müller cell marker GFAP, and a decrease in SOX2 gene expression (a marker for undifferentiated progenitor cells) compared to cells grown on unmodified tissue culture polystyrene (TCPS).

Retinal stem cell transplantation: Balancing safety and potential.

Stem cell transplantation holds great promise as a potential treatment for currently incurable retinal degenerative diseases that cause poor vision and blindness. Recently, safety data have emerged from several Phase I/II clinical trials of retinal stem cell transplantation. These clinical trials, usually run in partnership with academic institutions, are based on sound preclinical studies and are focused on patient safety. However, reports of serious adverse events arising from cell therapy in other poorly regulated centers have now emerged in the lay and scientific press. While progress in stem cell research for blindness has been greeted with great enthusiasm by patients, scientists, doctors and industry alike, these adverse events have raised concerns about the safety of retinal stem cell transplantation and whether patients are truly protected from undue harm. The aim of this review is to summarize and appraise the safety of human retinal stem cell transplantation in the context of its potential to be developed into an effective treatment for retinal degenerative diseases.

Advancing Clinical Trials for Inherited Retinal Diseases: Recommendations from the Second Monaciano Symposium.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.

Vitreous humor and albumin augment the proliferation of cultured retinal precursor cells.

Intravitreal injection is an important delivery route for studies involving the transplantation of various types of precursor cells to the retina; however, the effect on these cells of exposure to the vitreous microenvironment has not been specifically investigated. Here vitreous humor was evaluated for the potential to influence the proliferation of rat retinal precursor cells in vitro. Cells were isolated at embryonic day 19 and plated in standard proliferation medium in the presence or absence of fluid expressed from porcine vitreous humor. Cellular proliferation at different concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined as well. Results showed that addition of 20% vitreous fluid to primary rat retinal cultures significantly increased (3)H-thymidine incorporation compared with growth medium without vitreous supplementation. A vitreous fraction showing growth-promoting activity was localized to a molecular mass range <1000 Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors.

Sequential changes in the gene expression profile of murine retinal progenitor cells during the induction of differentiation.

PURPOSE: Following transplantation, cultured retinal progenitor cells (RPCs) integrate into the diseased host retina and exhibit morphologies and markers indicative of local cellular phenotypes. In vitro analysis of cultured RPCs allows detailed examination of marker gene expression during the initial phase of differentiation and can provide insight into the variables influencing this process. METHODS: Using cultured murine RPCs, this study compares the effects of fetal bovine serum (FBS) with those of ciliary neurotrophic factor (CNTF), individually or in combination with epidermal growth factor (EGF). Differentiation was assessed by way of the relative expression of 17 genes using quantitative PCR (qPCR) at five time points over a seven-day period. RESULTS: Both CNTF and FBS rapidly altered the gene expression of RPCs, with very marked upregulation of glial fibrillary acidic protein (GFAP; FBS>CNTF) and marked down-regulation of the proliferation marker K(i)-67, consistent with the induction of differentiation. The evidence supports a preponderantly pro-glial influence for both the FBS and CNTF, however, neuronal markers were also upregulated to a lesser extent. Immunocytochemistry confirmed subpopulations labeling with neuronal markers, including rhodopsin. In the presence of sustained EGF stimulation, the differentiating influences of both FBS and CNTF remained perceptible as transient peaks of relative gene expression, but were markedly diminished overall. CONCLUSIONS: This study shows that it is possible to compare the relative efficacy of in vitro differentiation protocols using murine RPCs and qPCR. The differentiating influences of both serum and CNTF were confirmed, but shown to be powerfully moderated by EGF. This suggests that EGF withdrawal is the dominant feature of these differentiation protocols and that exposure to either serum or CNTF is insufficient to irreversibly commit a cultured RPC population to terminal differentiation unless accompanied by concomitant cessation of mitogenic stimulation.

Growth kinetics and transplantation of human retinal progenitor cells.

We studied the growth kinetics of human retinal progenitor cells (hRPCs) isolated from donor tissue of different gestational ages (G.A.), determined whether hRPCs can be differentiated into mature photoreceptors and assessed their ability to integrate with degenerating host retina upon transplantation. Eyes (12-18 weeks G.A.) were obtained with IRB approval and retinas were enzymatically dissociated. Cells were expanded in vitro, counted at isolation and at each passage, and characterized using immunocytochemistry and PCR. GFP positive hRPCs were co-cultured with retinal explants from rd1 and rhodopsin -/- mice, or transplanted into B6 mice with retinal photocoagulation and rhodopsin -/- mice. Eyes were harvested for histological evaluation following transplantation. Our results show that hRPCs from 16 to 18 weeks G.A. had the longest survival in vitro and yielded the maximum number of cells, proliferating over at least 6 passages. These cells expressed the retinal stem cell markers nestin, Ki-67, PAX6 and Lhx2, and stained positively for photoreceptor markers upon differentiation with serum. Some of the GFP positive cells used for transplantation studies showed evidence of migration into the degenerative host retina and expressed rhodopsin. In conclusion, we have determined the growth kinetics of hRPCs and have shown that cells from donor tissue of 16-18 weeks G.A. exhibit the best proliferative dynamics under the specified conditions, and that hRPCs can also be differentiated along the photoreceptor lineage. Further, we have also demonstrated that following transplantation, some of these cells integrate within the host retina and differentiate to express rhodopsin, thereby supporting the potential utility of hRPC transplantation in the setting of retinal degenerative disorders.

Acute retinal ischemia caused by controlled low ocular perfusion pressure in a porcine model. Electrophysiological and histological characterisation.

The purpose of this study was to establish, and characterize a porcine model of acute, controlled retinal ischemia. The controlled retinal ischemia was produced by clamping the ocular perfusion pressure (OPP) in the left eye to 5 mm Hg for 2 h. The OPP was defined as mean arterial blood pressure (MAP) minus the intraocular pressure (IOP). It was clamped to 0-30 mm Hg by continuous monitoring of MAP and adjustment of the IOP, which was controlled by cannulation of the anterior chamber. Inner retinal function was assessed by induced multifocal electroretinography (mfERG) with comparisons of the amplitudes obtained in the experimental, left eye, and the control, right eye. Quantitative histology was performed to measure the survival of ganglion cells, amacrine cells and horizontal cells 2-6 weeks after the ischemic insult. An OPP of 5 mm Hg for 2h induced significant reductions in the amplitudes of iN1 to 20% (CI: 13-30%), and iP2 to 14% (95% CI: 8-22%) of their baseline values. No signs of recovery were found within the 6-week observation period. Quantitative histology revealed a highly significant reduction in the number of ganglion cells, amacrine cells and horizontal cells after the ischemic insult. This model seems to be suitable for investigations of therapeutic initiatives in diseases involving acute retinal ischemia.

Delayed administration of glial cell line-derived neurotrophic factor (GDNF) protects retinal ganglion cells in a pig model of acute retinal ischemia.

This study investigates whether intravitreal administration of glial cell line-derived neurotrophic factor (GDNF) enhances survival of NeuN positive retinal cells in a porcine model of retinal ischemia. 16 pigs were subjected to an ischemic insult where intraocular pressure was maintained at 5 mmHg below mean arterial blood pressure for 2 h. The mean IOP during the ischemic insult was 79.5 mmHg (s.e.m. 2.1 mmHg, n = 15). Three days after the insult the pigs received an intravitreal injection of GDNF microspheres or blank microspheres. The pigs were evaluated by way of multifocal electroretinography (mfERG), quantification of NeuN positive cells and evaluation of the degree of retinal perivasculitis and inflammation 6 weeks after the insult. In the post-injection eyes (days 14, 28 and 42), the ratios of the iN1 and the iP2 amplitudes were 0.10 (95% CI: 0.05-0.15) and 0.09 (95% CI: 0.04-0.16) in eyes treated with blank microspheres, and 0.24 (95% CI: 0.18-0.32) and 0.23 (95% CI: 0.15-0.33) in eyes treated with GDNF microspheres. These differences were statistically significant (P < 0.05). The number of NeuN positive cells in the area of the visual streak area was significantly higher in eyes injected with GDNF microspheres compared to eyes injected with blank microspheres. In eyes injected with GDNF microspheres the ganglion cell count was 9.5/field (s.e.m.: 2.1, n = 8), in eyes injected with blank microspheres it was 3.5/field (s.e.m.: 1.2, n = 7). This difference was statistically significant (P < 0.05). There was also a significant difference (P < 0.01) in the degree of perivasculiitis between GDNF treated eyes (median perivasculitis score 1.5) and blank treated eyes (median perivasculitis score 3.0). In conclusion, injection of GDNF microspheres 3 days after an ischemic insult results in functional and morphological rescue of NeuN positive cells in a porcine model of acute ocular ischemia.

CNS progenitor cells promote a permissive environment for neurite outgrowth via a matrix metalloproteinase-2-dependent mechanism.

Transplantation of progenitor cells to the CNS has shown promise in neuronal and glial replacement and as a means of rescuing host neurons from apoptosis. Here we examined the effect of progenitor grafts on neurite extension in the degenerating retina of rd1 (retinal degeneration 1) mice. Transplantation of retinal progenitor cells induced increased matrix metalloproteinase-2 (MMP2) secretion, partly from activated glial cells, which was then activated by neuronally expressed MMP14. Active MMP2 resulted in proteolysis of the neurite outgrowth inhibitors CD44 and neurocan in the degenerative retina, allowing significantly increased neurite outgrowth across the border between abutting nondystrophic and rd1 retinas. Progenitor-induced enhancement of outgrowth was abrogated by an MMP inhibitor or by coculture with retinal explants from MMP2-/- mice. This study provides the first identification of an MMP2-dependent mechanism by which exogenous progenitor cells alter the host environment to promote neural regeneration. This suggests a novel therapeutic role for progenitor cells in the treatment of CNS degenerative diseases.

Elevated MMP Expression in the MRL Mouse Retina Creates a Permissive Environment for Retinal Regeneration.

PURPOSE: The MRL/MpJ (healer) mouse is an established model for autoimmune studies and was recently identified as having a profound ability to undergo scarless regeneration of the tissue in the ear and heart. This regenerative capacity has been linked to elevated matrix metalloproteinase (MMP)-2 and -9 expression, giving this mouse the ability to degrade and remove inhibitory basement membrane molecules. Although elevated MMP expression has been reported in somatic tissues in this strain, little is known about MMP expression and the response to injury in the MRL/MpJ mouse retina. The purpose of this study was to investigate whether increased MMP expression and subsequent decreased inhibitory extracellular matrix molecule deposition in the MRL/MpJ mouse retina produces a permissive regenerative environment. METHODS: Experiments were performed using 3- to 4-week-old MRL/MpJ, retinal degenerative (rd1), and C57BL/6 (wild-type) mice. Western blotting, oligo-microarray, and immunohistochemical analyses were used to determine the level and location of MMP and extracellular matrix (ECM) protein expression. Retinal responses to injury were modeled by retinal detachment in vivo and in retinal explantation in vitro. The capacity of the retinal environment to support photoreceptor cell migration, integration, or regeneration was analyzed using hematoxylin-eosin, immunohistochemical staining, and cell counting. RESULTS: Compared with C57BL/6J animals, MRL/MpJ mice exhibit elevated levels of MMP-2, -9, and -14 and decreased levels of the inhibitory proteins neurocan and CD44 within the retina. Although similar increases in MMP-2, -9, and CD44s (CD44 degradation product) were observed in the rd1 retina, elevated levels of the inhibitory ECM molecules (neurocan and CD44) remained. Thus, the MRL retinal environment, which expresses lower levels of inhibitory ECM molecules after injury, was more conducive to regeneration and enhanced photoreceptor integration in vitro than C57BL/6J or rd1 controls. CONCLUSIONS: The MRL mouse retina shows elevated MMP expression and decreased levels of scar-related inhibitory molecules, which leads to a retinal environment that is more permissive for neural regeneration and cell integration after in vitro retinal explantation.

Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients.

Work in rodents has demonstrated that progenitor transplantation can achieve limited photoreceptor replacement in the mammalian retina; however, replication of these findings on a clinically relevant scale requires a large animal model. To evaluate the ability of porcine retinal progenitor cells to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP expression in subretinal grafts was high in cells expressing vimentin and lower in cells expressing photoreceptor markers, again consistent with possible downregulation during differentiation. Cells survived transplantation to the injured retina of allorecipients at all time points examined (up to 10 weeks) in the absence of exogenous immune suppression without indications of rejection. These findings demonstrate the feasibility of allogeneic progenitor transplantation in a large mammal and the utility of the pig in ocular regeneration studies.

A microfabricated scaffold for retinal progenitor cell grafting.

Diseases that cause photoreceptor cell degeneration afflict millions of people, yet no restorative treatment exists for these blinding disorders. Replacement of photoreceptors using retinal progenitor cells (RPCs) represents a promising therapy for the treatment of retinal degeneration. Previous studies have demonstrated the ability of polymer scaffolds to increase significantly both the survival and differentiation of RPCs. We report the microfabrication of a poly(glycerol-sebacate) scaffold with superior mechanical properties for the delivery of RPCs to the subretinal space. Using a replica molding technique, a porous poly(glycerol-sebacate) scaffold with a thickness of 45 microm was fabricated. Evaluation of the mechanical properties of this scaffold showed that the Young's modulus is about 5-fold lower and the maximum elongation at failure is about 10-fold higher than the previously reported RPC scaffolds. RPCs strongly adhered to the poly(glycerol-sebacate) scaffold, and endogenous fluorescence nearly doubled over a 2-day period before leveling off after 3 days. Immunohistochemistry revealed that cells grown on the scaffold for 7 days expressed a mixture of immature and mature markers, suggesting a tendency towards differentiation. We conclude that microfabricated poly(glycerol-sebacate) exhibits a number of novel properties for use as a scaffold for RPC delivery.

Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space.

Stem and progenitor cells can be combined with polymer substrates to generate tissue equivalents in culture. The replacement of retinal tissue lost to disease or trauma using retinal progenitor cells (RPCs) delivered on polymer scaffolds and transplanted into the sub-retinal space of the damaged retina is a promising therapeutic strategy. Micromachining-based, ultra-thin PMMA poly(methyl methacrylate) scaffolds may provide a suitable cytoarchitectural environment for tissue engineering and transplantation to the diseased eye. Here, adhesion of RPCs to polymer, as well as migration and differentiation in the host retina were compared for PMMA scaffolds (6 microm thickness) with either smooth or porous (11 microm diameter) surface topography. RPCs were cultured under identical conditions on smooth or porous laminin-coated polymer scaffolds and transplanted into the subretinal space of C57BL/6 mice. RPCs could be cultured on both scaffolds with similar results, although transplantation with non-porous scaffolds showed limited RPC retention. Porous scaffolds demonstrated enhanced RPC adherence during transplantation and allowed for greater process outgrowth and cell migration into the host retinal layers. Integrated cells expressed the mature neuronal marker neurofilament-200 (nf-200), the glial marker glial fibrillary acidic protein (GFAP) and the retinal-specific marker recoverin. No host foreign body response was seen. In conclusion, ultra-thin film PMMA scaffolds micromachined to contain through pores retain adherent RPCs to a considerably greater extent than unmachined versions during the transplantation process and can serve as a biocompatible substrate for cell delivery in vivo.

Intravitreal injections of GDNF-loaded biodegradable microspheres are neuroprotective in a rat model of glaucoma.

PURPOSE: To evaluate the efficacy of intravitreal injection of GDNF-loaded biodegradable microspheres in promoting the survival of retinal ganglion cells (RGCs) and their axons in a rat model of chronically elevated intraocular pressure (IOP). METHODS: Chronic elevation of IOP was induced in Brown Norway rats through injection of hypertonic saline (1.9 M) into the episcleral veins. After injection, IOP was measured twice a week in rats using topical anesthesia. Poly DL-lactide-co-glycolide (PLGA) microspheres were fabricated using a modified version of the spontaneous emulsification technique. Two and ten percent of volume solutions of microspheres loaded with glial cell line-derived neurotrophic factor (GDNF) were injected into the vitreous cavity of rats with elevated IOP, with injections of blank microspheres and PBS serving as controls. Histological analysis was used to quantify surviving RGCs and axons and provide comparison among different groups. In addition, the thickness of the retinal inner plexiform layer (IPL) and the level of glial fibrillary acidic protein (GFAP) expression within the retina and optic nerve were quantitatively compared. RESULTS: IOP was significantly increased in eyes with episcleral vein injection over untreated eyes (p<0.001) but did not show a significant difference among groups that received intravitreal injections of GDNF microspheres, blank microspheres, or PBS (p=0.1852). The duration of IOP elevation in this experiment was eight weeks. Expression of GDNF and its receptors localizes to the adult rat RGCs. Ten percent of the GDNF microsphere treatment significantly increased RGC survival and axon survival (p<0.001), reduced the loss of retinal IPL thickness (p<0.001), and decreased glial cell activation in the retina and optic nerve (p<0.001) compared with blank microspheres and PBS. In addition, GDNF microsphere treatment moderately reduced cupping of the optic nerve head. CONCLUSIONS: Delivery of GDNF via biodegradable microspheres significantly increased the survival of RGCs and their axons, preserved IPL thickness, and decreased retina and optic nerve glial cell activation in an experimental glaucoma model. This study suggests that GDNF delivered by PLGA microspheres may be useful as a neuroprotective tool in the treatment of glaucomatous optic neuropathy.