Growth Of Retinal Pigment Epithelial Cells Research Articles

Protective Effect of Curcumin on the Tight Junction Integrity and Cellular Senescence in Human Retinal Pigment Epithelium of Early Diabetic Retinopathy.

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells

Abstract Objectives Diabetic retinopathy (DR) is a retinal disease that arises from impaired glucose tolerance and leads to retinal microvascular leakages. Recent studies have indicated that DR pathogenesis is linked to dysfunctional retinal pigment epithelial (RPE) cells. Methods Investigating the potential interplay between endothelial cells (ECs) and RPE cells by treating ECs with high glucose (HG) and evaluating the function of cytokines released from ECs on the growth of RPE cells. Results The results revealed that high glucose-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) activated the NF-κB signaling pathway, increased intracellular levels of reactive oxygen species (ROS) and expression of caspase 3 while also elevating HUVECs delivery of cytokines such as VEGF, TNF-α, IL-6, and IL-1β. Conclusions As a result of our study, cytokines released from HG-treated HUVECs impede the growth of ARPE-19 in vitro, highlighting the importance of functional ECs for exploring the underlying mechanisms of vascular-associated retinal dysfunction. Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells.

Comparative analysis of the effects of limbal and adipose‐derived mesenchymal stem cell conditioned media on oxidative stress‐induced damage in retinal pigment epithelial cells

Aims/Purpose: The purpose of this investigation was to evaluate the impact of adipose and limbal tissue‐derived mesenchymal stem cell‐conditioned media on survival, and inflammation in H2O2‐induced retinal pigment epithelial cells.Methods: Following national regulations and with the approval of the Koç University Ethics and Investigation Committee, human limbal (L) and adipose‐derived (Ad) mesenchymal stem cells (MSCs) were isolated from cadaveric corneaoscleral rims from donors that originated from the cornea bank of Ministry of Health in Turkey and adipose tissue from discarded human liposuction surgery specimens, respectively. Conditioned medium (CM) that obtained by culturing MSCs in absence of serum was concentrated by filtration. Following a 24‐h pre‐treatment with H2O2, retinal pigment epithelial (RPE) cells were treated with MSC‐CM. Groups were defined as follows: Control, H2O2‐treatment (OS), OS‐Basal Medium, OS‐AdMSC‐CM and OS‐LMSC‐CM. Cell viability was assessed using MTT assay. Gene expression levels of pro‐ and anti‐inflammatory cytokines were analysed by qRT‐PCR.Results: Based on MTT assay, H2O2 inhibited the growth of RPE cells with IC50 values of 150 μM at 24 h. We found that MSC‐CM attenuated H2O2‐induced loss of cell viability. In comparison to the OS group, the LMSC‐CM and AdMSC‐CM treatments demonstrated significantly decreased levels of IL‐6, IL‐8, IL‐10 and TGF‐β1 gene expression.Conclusions: MSC‐CM ameliorated H2O2‐induced retinal pigment epithelial cell damage by alleviating inflammation.

Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Electrospun mini-MiSp spidroin/poly(L-lactide-co-ε-caprolactone) nanofibrous scaffolds for ARPE-19 cells

Age-related macular degeneration, a type of retinal degenerative disease, is the primary cause of severe visual impairment and irreversible blindness in people older than 50, due to age-related deterioration of the photoreceptors and the retinal pigment epithelium (RPE), which could be restored by cell therapy. However, the success of cell therapy depends on biocompatible scaffolds, which serve as carriers for the growth of RPE cells for repairing damaged or diseased retinas. In this study, the plasmid pET-SUMO-R2C for expressing a mini-spidroin, fusing the second repetitive region to the C-terminal domain of the minor ampullate spidroin gene from the spider Araneus ventricosus, was constructed and named R2C. The spidroin R2C was expressed, purified and wet spun into silk-like fibers, possessing a Young’s modulus of 3.51 ± 0.92 GPa and toughness of 24.67 ± 9.51 MPa. The spidroin R2C, mixed with poly(L-lactide-co-ε-caprolactone) (PLCL) at a ratio of 3:1, was electrospun into nanofibrous scaffolds, composed of nanometric fibers with a diameter of 751.70 ± 19.48 nm. After co-culturing with the R2C/PLCL scaffolds, the transcriptomic differences of the ARPE-19 cells were analyzed, showing that fourteen genes that are mainly related to inflammation, tumors and keratinization were upregulated more than three times, while seven genes that are mostly related to substance transport and homeostasis were downregulated more than three times. The R2C/PLCL scaffolds have the potential to be further studied to serve as a suitable carrier for RPE transplantation in the future.

Hydrogels to Support Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

Retinal pigment epithelial (RPE) cells are highly specialized neural cells with several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and, ultimately, blinding disease. While human embryonic stem cell-derived RPE cell (hESC-RPE) growth conditions are generally harsher than those of cell lines, the subretinal transplantation of hESC-RPE is being clinically explored as a strategy to recover the damaged retina and improve vision. The cell-adhesion ability of the support is required for RPE transplantation, where pre-polarized cells can maintain specific functions on the scaffold. This work examined four typical biodegradable hydrogels as supports for hESC-RPE growth. Four biodegradable hydrogels were examined: gelatin methacryloyl (GelMA), hyaluronic acid methacryloyl (HAMA), alginate, and fibrin hydrogels. ARPE-19 and hESC-RPE cells were seeded onto the hydrogels separately, and the ability of these supports to facilitate adherence, proliferation, and homogeneous distribution of differentiated hESC-RPE cells was investigated. Furthermore, the hydrogel's subretinal bio-compatibility was assessed in vivo. We showed that ARPE-19 and hESC-RPE cells adhered and proliferated only on the fibrin support. The monolayer formed when cells reached confluency, demonstrating the polygonal semblance, and revealing actin filaments that moved along the cytoplasm. The expression of tight junction proteins at cell interfaces on the 14th day of seeding demonstrated the barrier function of epithelial cells on polymeric surfaces and the interaction between cells. Moreover, the expression of proteins crucial for retinal functions and matrix production was positively affected by fibrin, with an increment of PEDF. Our in vivo investigation with fibrin hydrogels revealed high short-term subretinal biocompatibility. The research of stem cell-based cell therapy for retinal degenerative diseases is more complicated than that of cell lines. Our results showed that fibrin is a suitable scaffold for hESC-RPE transplantation, which could be a new grafting material for tissue engineering RPE cells.

Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue

Mesenchymal stromal cells (MSC) stop or slow retinal pigment epithelium (RPE) and neuroretina (NR) degeneration by paracrine activity in oxidative stress-induced retinal degenerative diseases. However, it is mandatory to develop adequate in vitro models that allow testing new treatment strategies against oxidative stress before performing in vivo studies. The viable double- and triple-layered setups are composed of separate layers of NR, MSC, and RPE (NR-MSC-RPE, NR-RPE, MSC-RPE) partially mimic in vivo retinal conditions. In this study, the paracrine neuroprotective effect of each setup's microenvironment on hydrogen peroxide (H2O2)-stressed was compared with unstressed RPE cells. RPE cell proliferation viability was assessed on day 1, 3, and 6 using Alamar Blue® (10%), MTT (10%) and a cell viability/cytotoxicity assay kit followed by data analysis. The results showed that RPE cells, highly viable (> 90%) in mixed medium of DMEM and neurobasal A (1:1), lost 50% viability on exposure to 400µM of H2O2 (P < 0.05). The unexposed groups differed significantly from exposed groups for RPE cell growth (RPE and [Formula: see text]RPE (P < 0.0001), NR-MSC-RPE, and NR-MSC-[Formula: see text]RPE (P < 0.05), NR-RPE and NR-[Formula: see text]RPE (P < 0.01), and MSC-RPE and MSC-[Formula: see text]RPE (P < 0.01). NR-[Formula: see text]RPE and NR-RPE supported RPE cell proliferation viability better than other setups (P < 0.01) and RPE cells proliferated 0.49-fold more in NR-MSC-[Formula: see text]RPE than NR-MSC-RPE. Thus, NR and MSC presence improved significantly each setup's microenvironment for cell rescue, nevertheless, each setup also showed limitations for its use as an in vitro study tool. Health of microenvironment of such setups depends on many factors including cell-secreted trophic factors.

HGF/Met Axis Protects Human Retinal Pigment Epithelial Cells from Cell Death

Demise of the retinal pigment epithelium (RPE) caused by etiologies such as age‐related macular degeneration is a major underlying factor leading to vision loss. Cell death can occur through two major pathways: apoptosis and necrosis/necroptosis. In the former, Fas/FasL death receptor signaling induces apoptosis, but its role in human RPE cell death is not thoroughly elucidated. In the latter, reactive oxygen species (ROS) can induce necrosis/necroptosis. Additionally, Hepatocyte Growth Factor (HGF) is a potent growth and survival factor for epithelial cells, but its functional role in RPE is not well characterized, especially its ability to protect RPE cells from death and to induce their growth. Accordingly, we hypothesized that HGF/Met protects human RPE cells from Fas‐promoted apoptosis and H2O2‐induced necrosis/necroptosis.We analyzed whether the HGF receptor (Met) is expressed in human and non‐human primate (NHP) eye tissue via western blot analysis and immunohistochemistry. To determine whether HGF/Met protects RPE cells against death induced by Fas/FasL and H2O2, we utilized gain‐ and loss‐of‐function models, cell survival assays and gene expression analysis (RNAseq). Human ARPE‐19 cell line was treated with various combinations of HGF, Met kinase inhibitor SU11274 (SU), FasL or H2O2. Cell growth and survival was determined via MTT assay. Met, Akt, and Erk activation were analyzed by western blot.Western blot analysis and immunohistochemistry confirmed Met activity and signaling in human and NHP eye tissue, including in the RPE cell layer. We found that HGF elicited Met stimulation and signaling culminating in activation of its downstream effectors Akt and Erk, proteins involved in two major pathways controlling cell survival and proliferation. We also uncovered that HGF inhibited FasL‐ and ROS‐induced killing of RPE. Conversely, inhibiting Met signaling rendered RPE susceptible to cell death induced by FasL and H2O2. Global gene expression analysis using an RNAseq approach revealed that HGF controls expression of numerous genes governing RPE cell growth, metabolism, and survival to name a few. In particular, HGF significantly upregulated 400 and repressed expression of 420 genes six hours post‐treatment; at 24 hours post‐treatment, HGF upregulated 291 and downregulated expression of 260 genes. Expression of 11,000 genes was unchanged.Our studies establish that HGF and Met play a major role in promoting the survival and homeostasis of RPE cells by inhibiting two major pathways of RPE cell death. Our findings suggest HGF/Met is a potential druggable target for therapeutic management of RPE‐related human ocular diseases.

Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth

Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1+ macrophages/microglia in human donor eye sections (aged and AMD) using multi-marker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD.

Inhibition of Obtusifolin on retinal pigment epithelial cell growth under hypoxia.

To explore the effect of Obtusifolin on retinal pigment epithelial cell growth under hypoxia. In vitro chemical hypoxia model of ARPE-19 cells was established using cobalt chloride (CoCl2). Cell viability was tested by cell counting kit-8 (CCK-8) assay. Western blot and real-time quantitative polymerase chain reaction were applied to detect proteins and mRNAs respectively. Flow cytometry was used to examine the cell cycle. Secretion of vascular endothelial growth factor (VEGF) was tested by using enzyme linked immunosorbent assay (ELISA). Under the chemical hypoxia model established by CoCl2, hypoxia inducible factor-1α (HIF-1α) mRNA and protein levels was up-regulated. Cell viability was increased and the proportion of S phase was higher. Obtusifolin could reduce cell viability under hypoxic conditions and arrest cells in G1 phase. Obtusifolin reduced the expression of Cyclin D1 and proliferating cell nuclear antigen (PCNA) in the hypoxic environment and increased the expression of p53 and p21. The levels of VEGF, VEGFR2 and eNOS proteins and mRNA were significantly increased under hypoxia while Obtusifolin inhibited the increasing. Obtusifolin can inhibit cell growth under hypoxic conditions and down-regulate HIF-1/VEGF/eNOS secretions in ARPE-19 cells.

The role of ADGRE5/CD97 in human retinal pigment epithelial cell growth and survival.

Cell surface molecules of retinal pigment epithelial (RPE) cells participate in the pathogenesis of retinal diseases. In an attempt to identify cell surface proteins that play a role in RPE cell-cell interactions, we have considered studying the expression, regulation, and signaling of ADGRE5/CD97, an adhesion G protein-coupled receptor family member, based on its known adhesive function in other cell types such as leukocytes. We showed that RPE cells express three isoforms of CD97 and identified inflammation-related cytokines as important mediators regulating CD97 expression. Whereas TNF-α and IFN-γ upregulated CD97, TGF-β decreased CD97 expression. Due to interaction with CD55, ARPE-19 cells firmly adhered to monocytes and T lymphocytes when overexpressing CD97, suggesting a role for CD97 in controlling leukocyte infiltration across the RPE-based blood-retinal barrier. CD97-mediated signaling acted synergistically with PDGF-BB and IFN-γ to regulate cell growth and survival, ensuring a cellular balance under inflammatory conditions. These findings suggest that CD97 on RPE cells serves to control leukocyte activation and trafficking in uveoretinal inflammation while at the same time regulating second messenger-mediated gene expression, cell growth, and survival.

Effects of the Macular Carotenoid Lutein in Human Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Oxidative stress-induced damage to the RPE occurs as part of the pathogenesis of age-related macular degeneration and neovascular retinopathies (e.g., retinopathy of prematurity, diabetic retinopathy). The xanthophyll carotenoids, lutein and zeaxanthin, are selectively taken up by the RPE, preferentially accumulated in the human macula, and transferred to photoreceptors. These macular xanthophylls protect the macula (and the broader retina) via their antioxidant and photo-protective activities. This study was designed to investigate effects of various carotenoids (β-carotene, lycopene, and lutein) on RPE cells subjected to either hypoxia or oxidative stress, in order to determine if there is effect specificity for macular pigment carotenoids. Using human RPE-derived ARPE-19 cells as an in vitro model, we exposed RPE cells to various concentrations of the specific carotenoids, followed by either graded hypoxia or oxidative stress using tert-butyl hydroperoxide (tBHP). The results indicate that lutein and lycopene, but not β-carotene, inhibit cell growth in undifferentiated ARPE-19 cells. Moreover, cell viability was decreased under hypoxic conditions. Pre-incubation of ARPE-19 cells with lutein or lycopene protected against tBHP-induced cell loss and cell co-exposure of lutein or lycopene with tBHP essentially neutralized tBHP-dependent cell death at tBHP concentrations up to 500 μM. Our findings indicate that lutein and lycopene inhibit the growth of human RPE cells and protect the RPE against oxidative stress-induced cell loss. These findings contribute to the understanding of the protective mechanisms attributable to retinal xanthophylls in eye health and retinopathies.

Odorant Receptor 51E2 Agonist β-ionone Regulates RPE Cell Migration and Proliferation.

The odorant receptor 51E2 (OR51E2), which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE) cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand β-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders.

Regulation of Matrix Metalloproteinase-2 Secretion from Scleral Fibroblasts and Retinal Pigment Epithelial Cells by miR-29a.

Purpose To identify an effective method to prevent myopia progression by characterizing the regulation of matrix metalloproteinase- (MMP-) 2 expression and its secretion from scleral fibroblasts and retinal pigment epithelium (RPE) cells by miR-29a. Methods The effects of miR-29a on the growth of scleral fibroblasts and RPE cells were assessed using the cell counting kit-8. The changes in MMP-2 mRNA levels in scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor were measured by quantitative PCR. Enzyme-linked immunosorbent assays were used to determine the changes in MMP-2 secretion from scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor. Results The miR-29a mimics or inhibitor did not significantly alter the growth of scleral fibroblasts or RPE cells at 24, 48, or 72 hours after transfection. MMP-2 mRNA levels were significantly decreased in scleral fibroblasts and RPE cells transfected with the miR-29a mimics. The secretion of MMP-2 by scleral fibroblasts and RPE cells was significantly decreased in cells transfected with the miR-29a mimics. Conclusions Suppression of scleral fibroblast and RPE cell expression and secretion of MMP-2 by miR-29a can be used as a therapeutic target for the prevention and treatment of myopia.

Blueberry Anthocyanin Promotes the Growth of Human Retinal Pigment Epithelial Cells

Blueberry Anthocyanin (BBA) has been proved to be beneficial to eyes and it could protect human Retinal Pigment Epithelial (RPE) cells, which perform essential functions for visual process, from light and oxidative damages. However, fundamental information about the effects of BBA on the growth of normal RPE cells is scarce. In the present study, the influences of BBA on the growth morphology, viability, cell cycle, Ki67 and PI3K/MAPK expression of RPE cells were investigated. RPE cells treated with 50 &mu g/mL BBA demonstrated a predominant polygonal morphology in mosaic arrangement, a better platform growing status, statistically higher viability, an increase in S-phase and more Ki67+cells. But neither pAkt nor pERK was detected in both groups. In conclusion, BBA could maintain high cell viability, boost DNA synthesis and preserve high percent of continuous cycling cells to promote cell survival and division without changing cellular morphology. The growth promotion effect of BBA might play a role in its damage-protective activities.

Cell delivery system using injectable polymeric films

Event Abstract Back to Event Cell delivery system using injectable polymeric films Hirokazu Kaji1, Taro Kondo1, Jin Suzuki1, Nobuhiro Nagai2 and Toshiaki Abe2 1 Tohoku University, Bioengineering and Robotics, Japan 2 Tohoku University, Clinical Cell Therapy, Japan Introduction: There have been ongoing efforts for the development of cell delivery system to overcome the intractable diseases. Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly population, whose main complication is the development of subretinal choroidal neovascularization and degeneration of retinal pigment epithelial (RPE) cells[1]. In this regard, subretinal transplantation of RPE cells to the degenerated site has attracted a great deal of attention as an innovative therapeutics for the treatment of AMD. However, poor viability, distribution and integration of the transplanted cells in suspension to the narrow subretinal space have limited this approach. Therefore, development of cell delivery system would bring significant benefits for the AMD treatment. Recently, we developed micropatterned ultrathin polymeric films (nanosheets), which can direct growth and morphogenesis of RPE cells toward local delivery of RPE cells[2]. Here, we examined whether micropatterned nanosheets can be delivered to the subretinal space of rat eyes using a syringe needle. Materials and Methods: Micropatterned nanosheets consisting of biodegradable poly(lactic-co-glycolic acid) (PLGA) and nile red were prepared on a poly(vinyl alcohol) (PVA) coated glass substrate by a combination of spincoating and microcontact printing technique. A typical diameter of circular nanosheets was 300 μm. The freestanding nanosheets were obtained by dissolving the PVA layer with PBS. Sprague-Dawley rats (SLC) weighing 250-300 g were used in this study. All animals were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research after receiving approval from the Institutional Animal Care and Use Committee of the Tohoku University Environmental & Safety Committee. After the rats were anesthetized with ketamine hydrochloride and xylazine hydrochloride, micropatterned nanosheets were injected into the subretinal space using a 27G syringe needle (Fig. 1a). Results and Discussion: Micropatterned nanosheets were injected with 2 μL of saline to the subretinal space through the sclera. Figure 1b shows optical coherence tomography (OCT) images of the retina taken one week after the injection of the micropatterned nanosheets. A shadow of the sheet like structure was observed in the subretinal space while there was not such shadow in the OCT image of a control. Also, a shadow of the circular nanosheet was observed at the posterior segment of the isolated eye (Fig. 1c). These results indicate that the nanosheets were successfully injected and spread into the subretinal space of rat eyes. Conclusions: We developed micropatterned nanosheets consisting of biodegradable polymers, which can be delivered to the subretinal space using a syringe needle. The injection of micropatterned nanosheets through a syringe needle could be a minimal invasive way to reduce the incision size of the sclera and subsequent inflammatory response. The flexible micropatterned nanosheets injectable by the syringe needle hold great promise to transplant organized RPE cells. Figure 1. (a) Schematic representation of the injection of micropatterned nanosheets into the subretinal space. (b) OCT images of (upper) the retina one week after the injection of the maicropatterned nanosheets and (lower) a control. Arrow in the upper image indicates the injected nanosheets. c) Photo of an isolated eyeball. Arrow indicates the injected nanosheets. JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (26286031); JSPS KAKENHI Grant-in-Aid for Challenging Exploratory Research (26560233); Japan National Society for the Prevention of Blindness

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

Improvement of RPE cells growth and metabolism abilities by mitochondrial transfer of neural stem cells

Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified. Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells. Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were co-cultured with NSCs, and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells, and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP, ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC). Results The third-generation of RPE cells grew well with the RPE65- and Bestrophin-positive rate >85%.The Mitotracker-red- labeled rates of NSCs and RPE cells were no less than 95%.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1phase and increased by 5% and 2% in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016, 0.114, 0.189). The contents of ATP, ADP and AMP in the RPE cells were (8.77±3.68), (2.76±0.92) and (1.07±0.65)μg/mg after cell co-culture, and those before co-culture were (11.29±2.29), (3.12±0.95) and (1.59±1.22)μg/mg, without significant differences between them (P=0.370, 0.668, 0.553). Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells. Key words: Stem cells; Pigment epithelium of eye/cytology; Retinal degeneration/therapy; Cells, cultured; Coculture techniques; Mitochondria/metabolism; Nanotubes; Animal

Development of a surface to increase retinal pigment epithelial cell (ARPE-19) proliferation under reduced serum conditions

Age related macular degeneration of the eye is brought about by damage to the retinal pigment epithelium (RPE) and is a major cause of adult blindness. One potential treatment method is transplantation of RPE cells grown in vitro. Maintaining RPE cell viability and physiological function in vitro is a challenge, and this must also be achieved using materials that can be subsequently used to deliver an intact cell sheet into the eye. In this paper, plasma polymerisation has been used to develop a chemically modified surface for maintaining RPE cells in vitro. Multiwell plates modified with a plasma copolymer of allylamine and octadiene maintained RPE cell growth at a level similar to that of TCPS. However, the addition of bound glycosaminoglycans (GAGs) to the plasma polymerised surface significantly enhanced RPE proliferation. Simply adding GAG to the culture media had no positive effect. It is shown that a combination of plasma polymer and GAG is a promising method for developing suitable surfaces for cell growth and delivery, that can be applied to any substrate material.

Bioactive substrates for human retinal pigment epithelial cell growth from elastin-like recombinamers

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruch's membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human.