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Microneedles (MNs) have recently garnered extensive interest concerning direct interstitial fluid (ISF) extraction or their integration into medical devices for continuous biomarker monitoring, owing to their advantages of painlessness, minimal invasiveness, and ease of use. However, micropores created by MN insertion may provide pathways for bacterial infiltration into the skin, causing local or systemic infection, especially with long-term in situ monitoring. To address this, we developed a novel antibacterial sponge MNs (SMNs@PDA-AgNPs) by depositing silver nanoparticles (AgNPs) on polydopamine (PDA)-coated SMNs. The physicochemical properties of SMNs@PDA-AgNPs were characterized regarding morphology, composition, mechanical strength, and liquid absorption capacity. The antibacterial effects were evaluated and optimized through agar diffusion assays in vitro. Wound healing and bacterial inhibition were further examined in vivo during MN application. Finally, the ISF sampling ability and biosafety of SMNs@PDA-AgNPs were assessed in vivo. The results demonstrate that antibacterial SMNs enable direct ISF extraction while preventing infection risks. SMNs@PDA-AgNPs could potentially be used for direct sampling or combined with medical devices for real-time diagnosis and management of chronic diseases.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.

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.

This paper presents a novel experiment teaching scheme for the control plane development of northbound applications in software defined networking (SDN). This scheme is designed to support the experiments of two commonly-used network applications, i.e., load balancing and network function virtualization. It is deployed on a virtual simulation experiment environment, which can be easily established in a timely manner. We have successfully integrated this experiment teaching scheme into the course of SDN in our university. With the support of this scheme, we not only provide students with meaningful practical guidance to learn northbound applications, but also help them better understand the programmable connotation of SDN. Furthermore, students can exploit the advantages of SDN to deploy new network services based on our proposed scheme, such that their ability of building practical and innovative network applications is enhanced.

According to the American Academy of Sleep Medicine (AASM), patients with apnea of 10 seconds or more , and more than 5 times per hour can be diagnosed with sleep disorders. The current gold standard for detecting this type of people with sleep disorder is Polysomnography (PSG). But more than 80% Obstructive Sleep Apnea(OSA) patients of the Sleep Disorder(SD) have not been detected. The reason is that the current configuration of PSG is insufficient in more than 2,500 sleep labs in the United States , and that the lack of manpower of sleep professional technicians who analyze PSG signal has caused many OSA patients to be diagnosed in a timely and effective manner. It is no effective measures to reduce the risk even after OSA patients have been diagnosed. Current medical treatments are either surgical or a lifelong Continuous positive dual channel air pressure ventilator(CPAP). Domestic research shows that OSA patients have poor sleep at night and sleepiness during the day. It often results in inefficient work and causes many traffic accidents. Therefore, how to take effective monitoring measures for these already diagnosed OSA patients has become an urgent problem to be solved.This paper extracts an interactive monitoring system for patients with OSA based on the Internet of Things(IoT) framework. It can reduce the timely rescue of OSA patients when they are in danger in field operations. At the same time, through the interactive function of this indicator mark, the anxiety during the waiting process can be reduced. It is also convenient for the peers to report the progress of the patient in time. The specific method is to use the existing IoT framework. The IoT data acquisition layer uses wearable sensors to collect vital signs of patients, with emphasis on ECG and SpO2 signals. The network layer transmits the collected physiological signals to the Beidou indicator using the Bluetooth Low Energy (BLE) protocol. The platform layer adopts the mature rescue interaction platform of Beidou&GPS. The previous GPS indicator has no short message function, and the patient can only passively wait for rescue. Positional standard is improved through Beidou model, and the short message interaction function has been added. Then the patient can report the progress of the disease in time while waiting for the rescue. After our simulation test, the effectiveness of the OSA patient rescue monitoring system based on the IoT framework has been greatly improved, especially in outdoor work, where the mobile phone signal coverage is relatively weak. The short message function added by the Beidou indicator can be used to provide timely progress of the patient's condition, and provide the medical rescue team with the data support to offer a more accurate rescue plan. After a comparative trial, the rescue rate of the OSA patients with the detection device of this article increased by 10 percentage points compared with the rescue rate with only GPS satellite phones.

Purpose To characterize histologic changes in the optic nerve and the retina of an end-stage retinitis pigmentosa (RP) patient after long-term implantation with the Argus II retinal prosthesis system. Methods Serial cross sections from the patient's both eyes were collected postmortem 6 years after implantation. Optic nerve from both eyes were morphometrically analyzed and compared. Retina underneath and outside the array was analyzed and compared with corresponding regions in the fellow eye. Results Although the optic nerve of the implant eye demonstrated significantly more overall atrophy than the fellow eye (P < 0.01), the temporal quadrant that retinotopically corresponded to the location of the array did not show additional damage. The total neuron count of the macular area was not significantly different between the two eyes, but the tack locations and their adjacent areas showed significantly fewer neurons than other perimacular areas. There was an increased expression of glial fibrillary acidic protein (GFAP) throughout the retina in the implant eye versus the fellow eye, but there was no significant difference in the cellular retinaldehyde-binding protein (CRALBP) expression. Except for the revision tack site, no significant increase of inflammatory reaction was detected in the implant eye. Conclusion Long-term implantation and electrical stimulation with an Argus II retinal prosthesis system did not result in significant tissue damage that could be detected by a morphometric analysis. Translational Relevance This study supports the long-term safety of the Argus II device and encourages further development of bioelectronics devices at the retina-machine interface.

PurposeTo create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats.MethodsFemale homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control.ResultsApproximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection.ConclusionsThis new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

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.

Multiple correlated phenotypes often appear in complex traits or complex diseases. These correlated phenotypes are useful in identifying gene-gene interactions associated with complex traits or complex disease more effectively. Some approaches have been proposed to use correlation among multiple phenotypes to identify gene-gene interactions that are common to multiple phenotypes. However these approaches either didn't find truly gene-gene interactions or got results which are hard to explain, especially by using all correlated phenotypes to identify gene-gene interactions, they made identified interactions unreliable. In this paper, we propose Multivariate Quantitative trait based Ordinal MDR (MQOMDR) algorithm to effectively identify gene-gene interactions associated with multiple correlated phenotypes by selecting the best classifier according to not only the training accuracy of the phenotype under consideration but also other phenotypes with weights determined mainly by their pair correlation with the phenotype under consideration and also by repeated selection process to make use of truly useful correlated phenotypes. Experimental results on two real datasets show that our algorithm has better performance in identifying gene-gene interactions associated with multiple correlated phenotypes.

One of the traditional ways for detecting dynamic communities is to find the communities at each interval through the static community detection algorithms. However, it usually leads to high computation complexity. In this paper, a novel algorithm based on the MapReduce model and the label propagation progress with the strategy of incremental related vertices is proposed, which is called PLPIRV (Parallel Label Propagation and Incremental Related Vertices). Based on the communities found at the previous interval, the new algorithm adjusts the communities the incremental related vertices belong to. The clustering of the whole network can be avoided by incrementally analyzing the variation of the networks, so that the time cost can be greatly reduced. Experiments on artificial and real datasets show that the proposed algorithm performs well on dynamic community detection. (Abstract).

The retinal pigmented epithelial (RPE) layer is one of the major ocular tissues affected by oxidative stress and is known to play an important role in the etiology of age-related macular degeneration (AMD), the major cause of blinding in the elderly. In the present study, sulindac, a nonsteroidal antiinflammatory drug (NSAID), was tested for protection against oxidative stress-induced damage in an established RPE cell line (ARPE-19). Besides its established antiinflammatory activity, sulindac has previously been shown to protect cardiac tissue against ischemia/reperfusion damage, although the exact mechanism was not elucidated. As shown here, sulindac can also protect RPE cells from chemical oxidative damage or UV light by initiating a protective mechanism similar to what is observed in ischemic preconditioning (IPC) response. The mechanism of protection appears to be triggered by reactive oxygen species (ROS) and involves known IPC signaling components such as PKG and PKC epsilon in addition to the mitochondrial ATP-sensitive K(+) channel. Sulindac induced iNOS and Hsp70, late-phase IPC markers in the RPE cells. A unique feature of the sulindac protective response is that it involves activation of the peroxisome proliferator-activated receptor alpha (PPAR-α). We have also used low-passage human fetal RPE and polarized primary fetal RPE cells to validate the basic observation that sulindac can protect retinal cells against oxidative stress. These findings indicate a mechanism for preventing oxidative stress in RPE cells and suggest that sulindac could be used therapeutically for slowing the progression of AMD.

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro , overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2–induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.

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.

A subretinal implant termed CPCB-RPE1 is currently being developed to surgically replace dystrophic RPE in patients with dry age-related macular degeneration (AMD) and severe vision loss. CPCB-RPE1 is composed of a terminally differentiated, polarized human embryonic stem cell-derived RPE (hESC-RPE) monolayer pre-grown on a biocompatible, mesh-supported submicron parylene C membrane. The objective of the present delivery study was to assess the feasibility and 1-month safety of CPCB-RPE1 implantation in Yucatan minipigs, whose eyes are similar to human eyes in size and gross retinal anatomy. This was a prospective, partially blinded, randomized study in 14 normal-sighted female Yucatan minipigs (aged 2 months, weighing 24–35 kg). Surgeons were blinded to the randomization codes and postoperative and post-mortem assessments were performed in a blinded manner. Eleven minipigs received CPCB-RPE1 while three control minipigs underwent sham surgery that generated subretinal blebs. All animals except two sham controls received combined local (Ozurdex™ dexamethasone intravitreal implant) and systemic (tacrolimus) immunosuppression or local immunosuppression alone. Correct placement of the CPCB-RPE1 implant was assessed by in vivo optical coherence tomography and post-mortem histology. hESC-RPE cells were identified using immunohistochemistry staining for TRA-1-85 (a human marker) and RPE65 (an RPE marker). As the study results of primary interest were nonnumerical no statistical analysis or tests were conducted. CPCB-RPE1 implants were reliably placed, without implant breakage, in the subretinal space of the minipig eye using surgical techniques similar to those that would be used in humans. Histologically, hESC-RPE cells were found to survive as an intact monolayer for 1 month based on immunohistochemistry staining for TRA-1-85 and RPE65. Although inconclusive regarding the necessity or benefit of systemic or local immunosuppression, our study demonstrates the feasibility and safety of CPCB-RPE1 subretinal implantation in a comparable animal model and provides an encouraging starting point for human studies.

Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays, respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C2 ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Over-expression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C2 ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late age-related macular degeneration lesions, and may spare the normal RPE monolayer. SphK1 over-expression increased cellular S1P, which promoted cell proliferation and protected RPE from ceramide-induced apoptosis. Understanding the relationship between the metabolism of sphingolipids and their effects in RPE cell survival/death may help us to develop effective and efficient therapies for retinal degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, targets the retinal pigment epithelium (RPE), a monolayer of cells at the back of the eye. As AMD progresses, it can develop into two distinct forms of late AMD: "dry," atrophic AMD, characterized by RPE senescence and geographic RPE loss, and "wet," neovascular AMD, characterized by RPE activation with abnormal growth of choroidal vessels. The genetic and molecular pathways that lead to these diverse phenotypes are currently under investigation. We have found that bone morphogenetic protein-4 (BMP4) is differentially expressed in atrophic and neovascular AMD. In atrophic AMD, BMP4 is highly expressed in RPE, and mediates oxidative stress induced RPE senescencein vitro via Smad and p38 pathways. In contrast, in neovascular AMD lesions, BMP4 expression in RPE is low, possibly a result of local expression of pro-inflammatory mediators. Thus, BMP4 may be involved in the molecular switch determining which phenotypic pathway is taken in the progression of AMD.

© 2015 Elsevier Ltd. Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. AMD is classified as either neovascular (NV-AMD) or non-neovascular (NNV-AMD). Cumulative damage to the retinal pigment epithelium, Bruch's membrane, and choriocapillaris leads to dysfunction and loss of RPE cells. This causes degeneration of the overlying photoreceptors and consequential vision loss in advanced NNV-AMD (Geographic Atrophy). In NV-AMD, abnormal growth of capillaries under the retina and RPE, which leads to hemorrhage and fluid leakage, is the main cause of photoreceptor damage. Although a number of drugs (e.g., anti-VEGF) are in use for NV-AMD, there is currently no treatment for advanced NNV-AMD. However, replacing dead or dysfunctional RPE with healthy RPE has been shown to rescue dying photoreceptors and improve vision in animal models of retinal degeneration and possibly in AMD patients. Differentiation of RPE from human embryonic stem cells (hESC-RPE) and from induced pluripotent stem cells (iPSC-RPE) has created a potentially unlimited source for replacing dead or dying RPE. Such cells have been shown to incorporate into the degenerating retina and result in anatomic and functional improvement. However, major ethical, regulatory, safety, and technical challenges have yet to be overcome before stem cell-based therapies can be used in standard treatments. This review outlines the current knowledge surrounding the application of hESC-RPE and iPSC-RPE in AMD. Following an introduction on the pathogenesis and available treatments of AMD, methods to generate stem cell-derived RPE, immune reaction against such cells, and approaches to deliver desired cells into the eye will be explored along with broader issues of efficacy and safety. Lastly, strategies to improve these stem cell-based treatments will be discussed.

Geographic atrophy (GA) in patients with age-related macular degeneration (AMD) is characterized by the loss of retinal pigment epithelial (RPE) cells in the macular region. Human embryonic stem cells (hESC)-derived RPE have shown promise in cellular therapy for these diseases. However, the differentiation from hESC is a very lengthy process with an initially low RPE yield. The purpose of this study was to determine whether polarized hESC-RPE conditioned medium (CM) could improve the yield of RPE differentiated from hESC and to identify the mechanism of this effect. Two hESC lines, hES3 and H9, were differentiated into RPE by culturing in CM or regular medium for up to 8 weeks. We found substantially more pigmented cells in CM based on surface area quantification, and Q-RT PCR showed significantly higher expression of RPE-specific genes in the H9 and hES3 cells cultured in CM. The results indicated that CM increased the yield of RPE cells differentiated from H9 and hES3 lines via increased RPE colony formation and proliferation. These findings may be beneficial to the manufacturing process for future clinical studies.

Oxidative stress-mediated injury to the retinal pigment epithelium (RPE) is a major factor involved in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Human embryonic stem cell (hESC)-derived RPE cells are currently being evaluated for their potential for cell therapy in AMD patients through subretinal injection of cells in suspension and subretinal placement as a polarized monolayer. To gain an understanding of how transplanted RPE cells will respond to the highly oxidatively stressed environment of an AMD patient eye, we compared the survival of polarized and nonpolarized RPE cultures following oxidative stress treatment. Polarized, nonpolarized/confluent, nonpolarized/subconfluent hESC-RPE cells were treated with H2O2. Terminal deoxynucleotidyl transferase dUTP nick end labeling stains revealed the highest amount of cell death in subconfluent hESC-RPE cells and little cell death in polarized hESC-RPE cells with H2O2 treatment. There were higher levels of proapoptotic factors (phosphorylated p38, phosphorylated c-Jun NH2-terminal kinase, Bax, and cleaved caspase 3 fragments) in treated nonpolarized RPE—particularly subconfluent cells—relative to polarized cells. On the other hand, polarized RPE cells had constitutively higher levels of cell survival and antiapoptotic signaling factors such as p-Akt and Bcl-2, as well as antioxidants superoxide dismutase 1 and catalase relative to nonpolarized cells, that possibly contributed to polarized cells' higher tolerance to oxidative stress compared with nonpolarized RPE cells. Subconfluent cells were particularly sensitive to oxidative stress-induced apoptosis. These results suggest that implantation of polarized hESC-RPE monolayers for treating AMD patients with geographic atrophy should have better survival than injections of hESC-RPE cells in suspension.

Retinal pigmented epithelium (RPE) secretes transforming growth factor beta 1 and 2 (TGF-β1 and -β2) cytokines involved in fibrosis, immune privilege, and proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be altered in various disease conditions including PVR and age-related macular degeneration, we determined levels of TGF-β from polarized human RPE (hRPE) and human stem cell derived RPE (hESC-RPE) as compared to nonpolarized cells. TGF-β2 was the predominant isoform in all cell culture conditions. Nonpolarized cells secreted significantly more TGF-β2 supporting the contention that loss of polarity of RPE in PVR leads to rise of intravitreal TGF-β2. Active TGF-β2, secreted mainly from apical side of polarized RPE, represented 6–10% of total TGF-β2. In conclusion, polarity is an important determinant of TGF-β2 secretion in RPE. Low levels of apically secreted active TGF-β2 may play a role in the normal physiology of the subretinal space. Comparable secretion of TGF-β from polarized hESC-RPE and hRPE supports the potential for hESC-RPE in RPE replacement therapies.

PURPOSE To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). METHODS Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). RESULTS The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. CONCLUSIONS hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.

Retina Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats Biju B. Thomas, 1,2 Danhong Zhu, 1,3 Li Zhang, 4 Padmaja B. Thomas, 5 Yuntao Hu, 6 Hossein Nazari, 1 Francisco Stefanini, 1 Paulo Falabella, 1 Dennis O. Clegg, 7 David R. Hinton, 1,3 and Mark S. Humayun 1,2 1 Department of Ophthalmology, USC Roski Eye Institute, University of Southern California, Los Angeles, California, United States USC Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, California, United States Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States Eye Center, Second Affiliated Hospital, Medical School of Zhejiang University, Hangzhou, China 5 Cellular Therapies Production Center, City of Hope, Duarte, California, United States Department of Ophthalmology, Beijing Tsinghua Changgung Hospital, Tsinghua University Medical Center, Beijing, China Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California, United States Correspondence: Biju B. Thomas, Department of Ophthalmology, Uni- versity of Southern California, Los Angeles, CA 90033, USA; biju.thomas@med.usc.edu. Submitted: January 28, 2016 Accepted: April 13, 2016 Citation: Thomas BB, Zhu D, Zhang L, et al. Survival and functionality of hESC-derived retinal pigment epitheli- um cells cultured as a monolayer on polymer substrates transplanted in RCS rats. Invest Ophthalmol Vis Sci. 2016;57:2877–2887. DOI:10.1167/ iovs.16-19238 P URPOSE . To determine the safety, survival, and functionality of human embryonic stem cell– derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. M ETHODS . Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n ¼ 46) received vitronectin-coated parylene membrane without cells (rMSPMþVN), group 2 (n ¼ 59) received rCPCB-RPE1 implants, and group 3 (n ¼ 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. R ESULTS . Eighty-seven percent of the rCPCB-RPE1–implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPMþVN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1–implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin- positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1– implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. C ONCLUSIONS . These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model. Keywords: transplantation, stem cells, parylene, retinal pigment epithelium, optokinetic testing, superior colliculus D ysfunction and death of RPE cells have been observed in human degenerative diseases leading to blindness, such as AMD. Age-related macular degeneration is one of the leading causes of blindness in the Western world. 1–3 Although antiangiogenic therapies were developed to treat exudative AMD, 4 there is no effective method for the treatment of dry AMD, particularly its end-stage, geographic atrophy. 5,6 A number of research groups are in pursuit of strategies to replace degenerated RPE cells with healthy RPE cells in the submacular space. 7–16 Various cell types have been examined for RPE cell replacement. These cells include immortalized cell lines, such as the human RPE cell line ARPE19, 17 sheets of adult RPE, 18 fetal RPE, 19 RPE derived from human embryonic stem cells (hESC-RPE), 20–24 human induced pluripotent stem cell–derived RPE, 9,25,26 and many non-RPE cell lines. 27–37 Stem cells, with their capacity to differentiate and replace diseased cells, can be an excellent source for RPE transplanta- tion. 10,11,16,28,29,38 Almost all previous studies used the method of cell suspension injection to evaluate the feasibility of stem cell–based RPE replacement therapies. This strategy is depen- dent on the integration of injected cells into the host RPE monolayer, which has been shown to occur in some studies 39 but not in others. 20 However, when injected as a cell suspension, most cells fail to form a polarized monolayer and survival rates are poor. 40,41 Furthermore, Bruch’s membrane, iovs.arvojournals.org j ISSN: 1552-5783 This work is licensed under a Creative Commons Attribution 4.0 International License. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/935270/ on 06/05/2017

This work reports a 3D parylene scaffold cage for culturing stem-cell-differentiated retinal pigment epithelial (RPE) cells for the therapy of age-related macular degeneration, which is caused by degenerated permeability of the Bruch's membrane first and then the loss of RPE cells. This reported cage can support sufficient nutrient permeation to nourish the RPE cells inside with in vivo like morphology. Compared to previous single layer scaffold substrates, this cage can effectively prevent the undesirable cell migration away from the scaffold.

Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.

Bone morphogenetic protein-4 (BMP4) may be involved in the molecular switch that determines which late form of age-related macular degeneration (AMD) an individual develops. BMP4 expression is high in retinal pigment epithelium (RPE) cells in late, dry AMD patients, while BMP4 expression is low in the wet form of the disease, characterized by choroidal neovascularization (CNV). Here, we sought to determine the mechanism by which BMP4 is down-regulated in CNV. BMP4 expression was decreased within laser-induced CNV lesions in mice at a time when tumor necrosis factor (TNF) expression was high (7 d postlaser) and was reexpressed in RPE when TNF levels declined (14 d postlaser). We found that TNF, an important angiogenic stimulus, significantly down-regulates BMP4 expression in cultured human fetal RPE cells, ARPE-19 cells, and RPE cells in murine posterior eye cup explants. We identified two specificity protein 1 (Sp1) binding sites in the BMP4 promoter that are required for basal expression of BMP4 and its down-regulation by TNF. Through c-Jun NH2-terminal kinase (JNK) activation, TNF modulates Sp1 phosphorylation, thus decreasing its affinity to the BMP4 promoter. The down-regulation of BMP4 expression by TNF in CNV and mechanisms established might be useful for defining novel targets for AMD therapy.—Xu, J., Zhu, D., He, S., Spee, C., Ryan, S. J., Hinton, D. R. Transcriptional regulation of bone morphogenetic protein 4 by tumor necrosis factor and its relationship with age-related macular degeneration.

This paper investigates the feasibility of a mesh-supported submicron-thick semipermeable parylene-C membrane as an artificial Bruch's membrane for the treatment of age-related macular degeneration (AMD). First, the permeabilities of parylene-C membranes with different thickness were measured and the results showed that as-deposited 0.30μm-thick parylene-C, which has a similar permeability to biological Bruch's membrane, is a good candidate to replace the diseased Bruch's membrane. This was further validated by perfusion cell culture experiments, in which nutrients were supplied to the cultured cells only by permeation through the submicron membrane. It was also found that retinal pigment epithelial (RPE) cells could grow and well-polarize with normal morphology on the membrane Finally, the implantation of the submicron-thick membrane (with in vitro cultured RPE cells) into rat's retina was successfully done with the help of a parylene-C/SU-8 microsurgical tool, and the following histological study of the retina showed that the reported thin parylene-C membrane could be a promissing RPE substrate candidate for further study.

The retinal pigment epithelium is a primary site of pathology in age-related macular degeneration. Oxidative stress and senescence are both thought to be important mediators of macular degeneration pathogenesis. We demonstrate here that bone morphogenetic protein-4 is highly expressed in the retinal pigment epithelium and adjacent extracellular matrix of patients with dry age-related macular degeneration. In vitro studies revealed that sublethal oxidative stress increased bone morphogenetic protein-4 expression in retinal pigment epithelial cells, and both bone morphogenetic protein-4 and persistent mild oxidative stress can induce retinal pigment epithelial cell senescence through p53-p21Cip1/WAF1-Rb pathway. We further demonstrate that bone morphogenetic protein-4 acts as a mediator in oxidative stress-induced senescence and that this mediator function is via Smad and the p38 signaling pathway to increase and activate p53 and p21Cip1/WAF1 and decrease phospho-Rb. Oxidative stress-induced senescence can be blocked by Chordin-like, an antagonist of bone morphogenetic protein-4, or SB203580, a phospho-p38 inhibitor. Our results suggest that oxidative stress and bone morphogenetic protein-4 may interact to promote retinal pigment epithelial cell senescence and that bone morphogenetic protein-4 may represent a novel therapeutic target to inhibit the progressive effects of oxidative stress and senescence in dry age-related macular degeneration.

Fibrosis plays an important role in the pathogenesis of several common blinding disorders, including proliferative diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and PVR;1–4 however, much needs to be learned about the basic pathophysiology of fibrosis in the intraocular environment.1 PVR may be viewed as a prototypical example of a protracted intraocular wound healing response that occurs when traction-generating cellular membranes develop in the vitreous and on the inner or outer surfaces of the retina following rhegmatogenous retinal detachment or major ocular trauma.5–7 RPE cells play a critical role in this epiretinal membrane formation8,9 These cells proliferate and migrate from the RPE monolayer to form sheets of dedifferentiated cells within a provisional extracellular matrix (ECM) containing fibronectin and thrombospondin.9–11 The protracted wound healing response causes the cellular membrane to become progressively more paucicellular and fibrotic.11 Experimental models of PVR have been developed to evaluate intraocular proliferation;12–16 however, these models typically exhibit cellular fibrinous strands without prominent fibrotic responses. Studies evaluating the role of those factors that elicit this fibrotic response are of particular interest. Normal ocular wound healing involves a tightly coordinated series of events: recruitment and activation of inflammatory cells, release of cytokines and growth factors, activation, proliferation and migration of ocular cells, secretion of extracellular matrix, tissue remodeling, and repair.1, 17 CTGF is an important stimulant of fibrosis,18 but its role in intraocular wound healing or PVR has not been studied in detail. CTGF is a secreted, cysteine-rich, heparin-binding polypeptide growth factor,19 20 that is rapidly upregulated after stimulation with serum or transforming growth factor-β (TGF-β). Various CTGF fragments have been shown to accumulate in tissue culture or body fluids while retaining their biologic activity.20–22 CTGF functions as a downstream mediator of TGF- β action on fibroblasts; it stimulates cell proliferation and cell matrix deposition (collagen 1 and fibronectin),18,20,23 and it may induce apoptosis.24,25 In addition to its action as a growth factor, CTGF has been implicated as an adhesive substrate in fibroblasts, mediated through α6β1 integrin.26 Importantly, CTGF is coordinately expressed with TGF-β and it demonstrates increased expression in numerous fibrotic disorders, including systemic sclerosis,27,28 pulmonary, renal, and myocardial fibrosis,29–32 and atherosclerosis.33 In the present study, we examine the process by which CTGF mediates the transformation of activated RPE into a fibrotic epiretinal membrane. Our results identify CTGF as a major mediator of retinal fibrosis and potentially an effective therapeutic target.

Human embryonic stem cell-derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs.The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium-derived factor (PEDF) levels from conditioned media.Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody.hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD.