Long-term Follow-up of a Phase 1/2a Clinical Trial of a Stem Cell-Derived Bioengineered Retinal Pigment Epithelium Implant for Geographic Atrophy.

PURPOSE: To report long-term results from a phase 1/2a clinical trial assessment of a scaffold-based human embryonic stem cell-derived retinal pigmented epithelium (RPE) implant in patients with advanced geographic atrophy (GA). DESIGN: A single-arm, open-label phase 1/2a clinical trial approved by the United States Food and Drug Administration. PARTICIPANTS: Patients were 69-85 years of age at the time of enrollment and were legally blind in the treated eye (best-corrected visual acuity [BCVA], ≤ 20/200) as a result of GA involving the fovea. METHODS: The clinical trial enrolled 16 patients, 15 of whom underwent implantation successfully. The implant was administered to the worse-seeing eye with the use of a custom subretinal insertion device. The companion nonimplanted eye served as the control. The primary endpoint was at 1 year; thereafter, patients were followed up at least yearly. MAIN OUTCOME MEASURES: Safety was the primary endpoint of the study. The occurrence and frequency of adverse events (AEs) were determined by scheduled eye examinations, including measurement of BCVA and intraocular pressure and multimodal imaging. Serum antibody titers were collected to monitor systemic humoral immune responses to the implanted cells. RESULTS: At a median follow-up of 3 years, fundus photography revealed no migration of the implant. No unanticipated, severe, implant-related AEs occurred, and the most common anticipated severe AE (severe retinal hemorrhage) was eliminated in the second cohort (9 patients) through improved intraoperative hemostasis. Nonsevere, transient retinal hemorrhages were noted either during or after surgery in all patients as anticipated for a subretinal surgical procedure. Throughout the median 3-year follow-up, results show that implanted eyes were more likely to improve by > 5 letters of BCVA and were less likely to worsen by > 5 letters compared with nonimplanted eyes. CONCLUSIONS: This report details the long-term follow-up of patients with GA to receive a scaffold-based stem cell-derived bioengineered RPE implant. Results show that the implant, at a median 3-year follow-up, is safe and well tolerated in patients with advanced dry age-related macular degeneration. The safety profile, along with the early indication of efficacy, warrants further clinical evaluation of this novel approach for the treatment of GA. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Peptide redesign for inhibition of the complement system: Targeting age-related macular degeneration.

PURPOSE: To redesign a complement-inhibiting peptide with the potential to become a therapeutic for dry and wet age-related macular degeneration (AMD). METHODS: We present a new potent peptide (Peptide 2) of the compstatin family. The peptide is developed by rational design, based on a mechanistic binding hypothesis, and structural and physicochemical properties derived from molecular dynamics (MD) simulation. The inhibitory activity, efficacy, and solubility of Peptide 2 are evaluated using a hemolytic assay, a human RPE cell-based assay, and ultraviolet (UV) absorption properties, respectively, and compared to the respective properties of its parent peptide (Peptide 1). RESULTS: The sequence of Peptide 2 contains an arginine-serine N-terminal extension (a characteristic of parent Peptide 1) and a novel 8-polyethylene glycol (PEG) block C-terminal extension. Peptide 2 has significantly improved aqueous solubility compared to Peptide 1 and comparable complement inhibitory activity. In addition, Peptide 2 is more efficacious in inhibiting complement activation in a cell-based model that mimics the pathobiology of dry AMD. CONCLUSIONS: We have designed a new peptide analog of compstatin that combines N-terminal polar amino acid extensions and C-terminal PEGylation extensions. This peptide demonstrates significantly improved aqueous solubility and complement inhibitory efficacy, compared to the parent peptide. The new peptide overcomes the aggregation limitation for clinical translation of previous compstatin analogs and is a candidate to become a therapeutic for the treatment of AMD.

Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration.

We introduce a human retinal pigmented epithelial (RPE) cell-culture model that mimics several key aspects of early stage age-related macular degeneration (AMD). These include accumulation of sub-RPE deposits that contain molecular constituents of human drusen, and activation of complement leading to formation of deposit-associated terminal complement complexes. Abundant sub-RPE deposits that are rich in apolipoprotein E (APOE), a prominent drusen constituent, are formed by RPE cells grown on porous supports. Exposure to human serum results in selective, deposit-associated accumulation of additional known drusen components, including vitronectin, clusterin, and serum amyloid P, thus suggesting that specific protein-protein interactions contribute to the accretion of plasma proteins during drusen formation. Serum exposure also leads to complement activation, as evidenced by the generation of C5b-9 immunoreactive terminal complement complexes in association with APOE-containing deposits. Ultrastructural analyses reveal two morphologically distinct forms of deposits: One consisting of membrane-bounded multivesicular material, and the other of nonmembrane-bounded particle conglomerates. Collectively, these results suggest that drusen formation involves the accumulation of sub-RPE material rich in APOE, a prominent biosynthetic product of the RPE, which interacts with a select group of drusen-associated plasma proteins. Activation of the complement cascade appears to be mediated via the classical pathway by the binding of C1q to ligands in APOE-rich deposits, triggering direct activation of complement by C1q, deposition of terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular aspects of AMD pathogenesis, and the testing of new therapeutic agents for its treatment.

Anti-amyloid therapy protects against retinal pigmented epithelium damage and vision loss in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid ß (Aß) peptides, Aß1-40 (Aß40) and Aß1-42 (Aß42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aß, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aß-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aß40 and Aß42. Concomitant reduction in the levels of Aß and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aß40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aß antibodies. They also implicate Aß in the pathogenesis of AMD and identify Aß as a viable therapeutic target for its treatment.

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.

A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.

OBJECTIVE: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. METHODS: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. RESULTS: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat's subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. CONCLUSION: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat's subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

Microcarrier-Based Culture of Human Pluripotent Stem-Cell-Derived Retinal Pigmented Epithelium.

Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.

Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration.

Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34+ structures beneath the implant and CD4+, CD68+, and FoxP3+ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study. Subretinally implanted, HLA-mismatched donor RPE cells survive, express functional markers, and do not elicit clinically detectable intraocular inflammation or serologic immune responses even without long-term immunosuppression.

Memory in induced pluripotent stem cells: reprogrammed human retinal-pigmented epithelial cells show tendency for spontaneous redifferentiation.

Induced pluripotent stem (iPS) cells have been generated from a variety of somatic cell types via introduction of transcription factors that mediate pluripotency. However, it is unknown that all cell types can be reprogrammed and whether the origin of the parental cell ultimately determines the behavior of the resultant iPS cell line. We sought to determine whether human retinal-pigmented epithelial (RPE) cells could be reprogrammed, and to test the hypothesis that reprogrammed cells retain a "memory" of their origin in terms of propensity for differentiation. We reprogrammed primary fetal RPE cells via lentiviral expression of OCT4, SOX2, LIN28, and Nanog. The iPS cell lines derived from RPE exhibited morphologies similar to human embryonic stem cells and other iPS cell lines, expressed stem cell markers, and formed teratomas-containing derivatives of all three germ layers. To test whether these iPS cells retained epigenetic imprints from the parental RPE cells, we analyzed their propensity for spontaneous differentiation back into RPE after removal of FGF2. We found that some, but not all, iPS lines exhibited a marked preference for redifferentiation into RPE. Our results show that RPE cells can be reprogrammed to pluripotency, and suggest that they often retain a memory of their previous state of differentiation.

One-Year Follow-Up in a Phase 1/2a Clinical Trial of an Allogeneic RPE Cell Bioengineered Implant for Advanced Dry Age-Related Macular Degeneration.

Purpose: To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD). Methods: The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results. Results: The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6-13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss (47% vs. 33%; P = not significant). Conclusions: Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD. Translational Relevance: This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD.

Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin alpha v beta 5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential.

Accumulation of extracellular RGR-d in Bruch's membrane and close association with drusen at intercapillary regions.

Human retinal pigment epithelial (RPE) cells synthesize an extraneous splice isoform of retinal G protein-coupled receptor (RGR). In this study, we analyzed the exon-skipping variant of RGR (RGR-d) that is found in extracellular deposits. RPE-choroid tissue sections were prepared from postmortem human eyes from donors of various ages. RGR-d was analyzed in drusen and Bruch's membrane by immunohistochemical localization. Extracellular RGR-d is present in most drusen, including hard, soft, confluent and early-stage. Initial drusen formation is known to be preferentially associated with the intercapillary regions of Bruch's membrane. We corroborated this significant association of drusen, including early-stage drusen, with the intercapillary regions. The distribution of extracellular RGR-d in Bruch's membrane differs in old and young donors. In older persons, nodes of concentrated RGR-d accumulate at intercapillary loci, predominantly at the lateral edges of the capillaries of the choriocapillaris. RGR-d loci at the lateral capillary wall appear numerous in old, but not young, donors. Intensely immunostained RGR-d loci can be found at the base of early-stage drusen mounds in the older donors and may precede the formation of these drusen.

Complement factor H in AMD: Bridging genetic associations and pathobiology.

Age-Related Macular Degeneration (AMD) is a complex multifactorial disease characterized in its early stages by lipoprotein accumulations in Bruch's Membrane (BrM), seen on fundoscopic exam as drusen, and in its late forms by neovascularization ("wet") or geographic atrophy of the Retinal Pigmented Epithelial (RPE) cell layer ("dry"). Genetic studies have strongly supported a relationship between the alternative complement cascade, in particular the common H402 variant in Complement Factor H (CFH) and development of AMD. However, the functional significance of the CFH Y402H polymorphism remains elusive. In this article, we critically review the literature surrounding the functional significance of this polymorphism. Furthermore, based on our group's studies we propose a model in which CFH H402 affects CFH binding to heparan sulfate proteoglycans leading to accelerated lipoprotein accumulation in BrM and drusen progression. We also review the literature on the role of other complement components in AMD pathobiologies, including C3a, C5a and the membrane attack complex (MAC), and on transgenic mouse models developed to interrogate in vivo the effects of the CFH Y402H polymorphism.

A bioengineered retinal pigment epithelial monolayer for advanced, dry age-related macular degeneration.

Retinal pigment epithelium (RPE) dysfunction and loss are a hallmark of non-neovascular age-related macular degeneration (NNAMD). Without the RPE, a majority of overlying photoreceptors ultimately degenerate, leading to severe, progressive vision loss. Clinical and histological studies suggest that RPE replacement strategies may delay disease progression or restore vision. A prospective, interventional, U.S. Food and Drug Administration-cleared, phase 1/2a study is being conducted to assess the safety and efficacy of a composite subretinal implant in subjects with advanced NNAMD. The composite implant, termed the California Project to Cure Blindness-Retinal Pigment Epithelium 1 (CPCB-RPE1), consists of a polarized monolayer of human embryonic stem cell-derived RPE (hESC-RPE) on an ultrathin, synthetic parylene substrate designed to mimic Bruch's membrane. We report an interim analysis of the phase 1 cohort consisting of five subjects. Four of five subjects enrolled in the study successfully received the composite implant. In all implanted subjects, optical coherence tomography imaging showed changes consistent with hESC-RPE and host photoreceptor integration. None of the implanted eyes showed progression of vision loss, one eye improved by 17 letters and two eyes demonstrated improved fixation. The concurrent structural and functional findings suggest that CPCB-RPE1 may improve visual function, at least in the short term, in some patients with severe vision loss from advanced NNAMD.

Synaptic pathology, altered gene expression, and degeneration in photoreceptors impacted by drusen.

PURPOSE: Drusen are risk factors for age-related macular degeneration and have been shown to negatively impact cells of the RPE and retina. In this study, the effects of drusen on the synaptic machinery of retinal photoreceptors are investigated. METHODS: Human donor eye tissue containing retina, RPE, and choroid was processed for confocal immunofluorescence microscopy, laser capture microdissection, and light and electron microscopy. Tissue sections were immunostained with a panel of antibodies to synapse-associated proteins. Populations of photoreceptors over drusen and normal populations of photoreceptors were microdissected from fresh frozen tissue, RNA was purified, and quantitative PCR was performed to compare relative levels of gene expression. RESULTS: The number of photoreceptor synaptic terminals is reduced in regions of the outer plexiform layer over drusen, synaptic proteins are mislocalized in photoreceptor cells, and synaptic terminals are often observed within the outer nuclear layer. Photoreceptors over drusen also increase expression of the stress response proteins apolipoprotein E and alphaB-crystallin. Abnormal immunolabeling patterns are not restricted to photoreceptors directly over drusen but are also observed in cells flanking drusen. Gene expression analysis confirms reductions in the expression of genes coding for synapse-associated proteins and signal transduction proteins and increases in the expression of apolipoprotein E and alphaB-crystallingene transcripts. Ultrastructural analysis of photoreceptor synaptic terminals over drusen reveals significant abnormalities, and cell counts show a reduction in photoreceptor density directly over, and lateral to, drusen of all sizes. CONCLUSIONS: Photoreceptors overlying and flanking drusen exhibit morphologic and biochemical signs of degeneration. The expression of synapse-associated proteins decreases in photoreceptor synaptic terminals, whereas the expression of stress-response proteins increases. Reductions in photoreceptor cell densities over, and flanking, drusen suggest that these degenerative effects eventually result in the death of photoreceptors.

Cellular models and therapies for age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex neurodegenerative visual disorder that causes profound physical and psychosocial effects. Visual impairment in AMD is caused by the loss of retinal pigmented epithelium (RPE) cells and the light-sensitive photoreceptor cells that they support. There is currently no effective treatment for the most common form of this disease (dry AMD). A new approach to treating AMD involves the transplantation of RPE cells derived from either human embryonic or induced pluripotent stem cells. Multiple clinical trials are being initiated using a variety of cell therapies. Although many animal models are available for AMD research, most do not recapitulate all aspects of the disease, hampering progress. However, the use of cultured RPE cells in AMD research is well established and, indeed, some of the more recently described RPE-based models show promise for investigating the molecular mechanisms of AMD and for screening drug candidates. Here, we discuss innovative cell-culture models of AMD and emerging stem-cell-based therapies for the treatment of this vision-robbing disease.

Restoration of mesenchymal retinal pigmented epithelial cells by TGFß pathway inhibitors: implications for age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD. METHODS: Transcriptome wide expression profiles of human fetal RPE cell cultures as a function of passage and time post-plating were determined using Agilent 44 K whole genome microarrays and RNA-Seq. Using a systems level analysis, differentially expressed genes and pathways of interest were identified and their role in the establishment of a persistent mesenchymal state was assessed using pharmacological-based experiments. RESULTS: Using a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we show that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40 % of the transcriptome. In contrast, at subconfluence fewer than 5 % of expressed transcripts have two-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFß pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and largely reverses the passage-dependent loss of epithelial potential; thus extending the effective lifespan by at least four passages. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD, including key members of the TGFß pathway. CONCLUSIONS: In RPE cells the switch to a persistent mesenchymal state following prolonged wound stimulus is driven by lasting activation of the TGFß pathway. Targeted inhibition of TGFß signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell-based therapies.

New compstatin peptides containing N-terminal extensions and non-natural amino acids exhibit potent complement inhibition and improved solubility characteristics.

Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases.

Drusen-associated degeneration in the retina.

PURPOSE: Drusen are variably sized extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. They are commonly found in aged eyes, however, numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration. The purpose of this study was to investigate the impact of drusen on overlying cells of the retina. METHODS: Tissue containing retina and RPE/choroid was dissected from human donor eyes, embedded in agarose, and sectioned at 100 micro m using a vibratome. Sections were immunostained with a panel of antibodies that labeled glial cells, first-, second-, and third-order retinal neurons and processed for confocal microscopy. RESULTS: Retinal cells that overlie both soft and hard drusen exhibited numerous structural and molecular abnormalities. Normally detectable only in the outer segments of rod photoreceptors, rod opsin immunolabeling was also observed in the inner segment, cell body, axon, and axon terminal of photoreceptors that overlie drusen. Labeling with this antibody also revealed the deflection and shortening of rod inner and outer segments. Cone photoreceptors displayed similar structural abnormalities, as well as a decrease in cone opsin immunoreactivity. Drusen-associated abnormalities in the synaptic terminals of photoreceptor cells were also observed. In addition, an increase in intermediate filament protein immunoreactivity (vimentin and glial fibrillary acidic protein) was observed within Müller glial cells in areas of retina overlying drusen. Both soft and hard drusen were associated with a similar spectrum of effects in both macular and extramacular regions. Second- and third-order neurons, including bipolar, horizontal, amacrine, and ganglion cells all appeared unaffected. The structural and molecular abnormalities observed in photoreceptors and Müller glial cells were confined to retinal regions directly overlying and immediately adjacent to drusen; more distant retinal regions appeared unperturbed. Remarkably, significant abnormalities were observed over small subclinical drusen. CONCLUSIONS: Retinal cells overlying both soft and hard drusen exhibit structural and molecular abnormalities indicative of photoreceptor degeneration and Müller glial activation. These abnormalities resemble the degenerative effects common to many forms of retinal degeneration, but are confined to areas directly overlying drusen. This suggests that photoreceptor cell function is compromised as a consequence of drusen formation.