Cell-derived Retinal Pigment Epithelial Cells Research Articles

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration.In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. Statement of significanceArtificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.

A Preclinical Safety Study of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells for Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

This issue of Acta

This issue of Acta

Porous polybutylene succinate films enabling adhesion of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE)

In the last decade, several studies have shown that polybutylene succinate (PBSu) has a high potential as a biomaterial enabling cell adhesion and growth. In this study, porous PBSu films have been prepared by the breath figure method (BF) and by particulate leaching (PL), and characterized in terms of thickness, surface properties, diffusion capacity and in vitro stability. Because porous films are of high interest for tissue engineering of retinal pigment epithelium (RPE), the initial viability and adhesion of human embryonic stem cell-derived RPE onto the PBSu films was then evaluated. To the best of our knowledge, this is the first study on the adhesion behavior of hESC-RPE onto porous and biodegradable polymer surfaces. The results clearly demonstrated that the two manufacturing methods produced materials with very distinct properties. Films produced by BF expressively demonstrated the highest roughness and surface area, and the lowest water contact angle. These features likely contributed to increase the biocompatibility of the surface, particularly when coated with laminin and collagen IV, as observed by the improved cell viability, cell morphology, adhesion and production of extracellular matrix proteins. Altogether, our results showed not only that PBSu holds high potential in retinal tissue engineering, but also that the physical properties and biocompatibility of the material are highly dependent on the adopted casting method.

Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine.

Regenerative medicine has received a lot of attention as a novel strategy for injuries and diseases that are difficult to cure using current techniques. Cell production, which is vital for regenerative medicine, has undergone remarkable progress via breakthroughs in developmental biology and tissue engineering; currently, cell production requires numerous experimental operators performing manual, small-scale cell cultures. Other major obstacles for cell production and regenerative medicine include the variable quality of products based on the experimental procedure, the skills of operators, the level of labor required for production, and costs. Technological developments are required to overcome this, including automation instead of manual culture. Age-related macular regeneration (AMD) is a refractory ocular disease that causes severe deterioration in central vision due to senescence in the retinal pigment epithelium (RPE). Recently, we performed an autologous transplantation of induced pluripotent stem (iPS) cell-derived RPE cell sheets and started clinical research on allografts from RPE cell suspensions differentiated from iPS cells. The use of regenerative therapies for AMD using iPS cell-derived RPE is expected to become more widespread. In the present study, human iPS cell-derived RPE cells were cultured to form RPE cell sheets using equipment with a closed culture module. The quality of the automated cultured RPE cell sheets was confirmed by comparing their morphological and biological properties with those of manually generated RPE cell sheets. As a result, machine-cultured RPE sheets displayed the same quality as manually cultured RPE sheets, showing that iPS cell-derived RPE cell sheets were successfully cultured by an automated process.

Critical Functionality Effects from Storage Temperature on Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Suspensions

Human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (hiPSC-RPE) cells suspension have the potential for regenerative treatment. However, practical regenerative applications with hiPSC-RPE cells require the development of simple and cost-effective non-freezing preservation methods. We investigated the effect of non-freezing temperatures on suspended hiPSC-RPE cells in various conditions and analysed mechanisms of cell death, anoikis, Rho GTPases, hypoxia, microtubule destruction, and cell metabolism. Cells stored at 37 °C had the lowest viability due to hypoxia from high cell metabolism and cell deposits, and cells preserved at 4 °C were damaged via microtubule fragility. Cell suspensions at 16 °C were optimal with drastically reduced apoptosis and negligible necrosis. Moreover, surviving cells proliferated and secreted key proteins normally, compared to cells without preservation. hiPSC-RPE cell suspensions were optimally preserved at 16 °C. Temperatures above or below the optimal temperature decreased cell viability significantly yet differentially by mechanisms of cell death, cellular metabolism, microtubule destruction, and oxygen tension, all relevant to cell conditions. Surviving cells are expected to function as grafts where high cell death is often reported. This study provides new insight into various non-freezing temperature effects on hiPSC-RPE cells that are highly relevant to clinical applications and may improve cooperation between laboratories and hospitals.

Survival and functionality of xeno-free human embryonic stem cell-derived retinal pigment epithelial cells on polyester substrate after transplantation in rabbits.

To study immunogenic properties of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis invitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE±interferon-gamma (IFN-γ) stimulation (n=1). Xeno-free hESC-RPE on PET with TER<200Ω·cm2 >or PET alone were transplanted into 18 rabbits with short-term triamcinolone±extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4weeks, the eyes were processed for histology and transmission electron microscopy. Upon invitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n=1). Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.

Screening and optimization of potential injection vehicles for storage of retinal pigment epithelial stem cell before transplantation.

Retinal pigment epithelial (RPE) cells are highly specialized neural cells that have several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and ultimately the blinding disease age-related macular degeneration. Subretinal transplantation of stem cell-derived RPE cell suspensions is being explored as a strategy to recover the damaged retina and improve vision. This approach may be improved by developing a vehicle that increases postinjection cell viability and distribution and integration of RPE cells. In this study, Food and Drug Administration-approved natural polymers, including alginate, methylcellulose, and hyaluronic acid (HA), were examined for performance as cell vehicles for adult human RPE stem cells (RPESCs). We compared the effect of RPESC storage as a cell suspension in these delivery vehicles for 1-96hr at different temperatures on subsequent cell performance in a cell culture model. RPESC survival, attachment, distribution, proliferation, and differentiation into RPE cells were monitored by microscopy over the course of 8weeks. Our in vitro results demonstrate that RPESC suspension in a 0.2% HA solution promotes better initial cell distribution, proliferation, cobblestone formation, and expression of RPE cell markers (microphthalmia-associated transcription factor and orthodenticle homeobox 2) after 96hr of storage. These data suggest that HA addition to the vehicle can significantly enhance RPESC performance in vitro and is a promising strategy to pursue an improved delivery vehicle supporting in vivo RPE cell transplantation.

Detection of Complement Activators in Immune Attack Eyes After iPS-Derived Retinal Pigment Epithelial Cell Transplantation.

To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can express complement factors. To confirm expression of complement factors in human iPS-RPE cells, we performed flow cytometry, immunohistochemistry, ELISA, and quantitative RT-PCR for the following: C3, C5, CFB (Factor B), C5b-9 (membrane attack complex [MAC]), CFH (Factor H), CFI (Factor I), CD46, CD55, CD59, clusterin, and vitronectin. We also prepared iPS-RPE cells in the presence of recombinant IFN-γ, recombinant TNF-α, lipopolysaccharide, supernatants of naïve T cells, and T helper 1 (Th1) cells. For the transplantation, after preparation of iPS-RPE cells from cynomolgus monkeys, the iPS-RPE cells (allografts) were transplanted into the subretinal space in monkeys. After surgery, monkeys were euthanized for IHC evaluation of the retinal section and determination of complement factors (C3, C5, CFB, MAC, and C1q), cytokines, and immunoglobulin G (IgG). Human iPS-RPE cells expressed complement activators and inhibitors. iPS-RPE cells highly expressed complement factors during inflammatory conditions, especially IFN-γ exposure including Th1 cell supernatants. In immune attack eyes after allogeneic iPS-RPE cell transplantation, complement activators such as C3, CFB, C5, and MAC were detected around the host RPE layer, grafted RPE cells, inflammatory retinal lesions, and transplanted subretinal space. In addition, we observed a large number of C1q and IgG double positive and IFN-γ positive inflammatory cells in the retinal sections. iPS-derived RPE cells greatly expressed complement factors. Thus, RPE cells might be activated and produce complement factors after exposure to infiltrating inflammatory cells in the eye.

Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

SummaryAge-related macular degeneration is caused by dysfunction and loss of retinal pigment epithelium (RPE) cells, and their transplantation may rescue visual functions and delay disease progression. Human embryonic stem cells (hESCs) may be an unlimited source of RPE cells for allotransplantation. We analyzed the immunomodulatory properties of hESC-derived RPE (hESC-RPE) cells, and showed that they inhibited T cell responses. Co-culture experiments showed that RPE cells inhibited interfon-γ secretion and proliferation of activated T cells. Furthermore, hESC-RPE cells enhanced T cell apoptosis and secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). In addition, RPE cells altered the expression of T cell activation markers, CD69 and CD25. RPE cells transplanted into RCS rats without immunosuppression survived, provided retinal rescue, and enhanced IL-10 blood levels. Our data suggest that hESC-RPE cells have immunosuppressive properties. Further studies will determine if these properties are sufficient to alleviate the need for immunosuppression therapy after their clinical allotransplantation.

A cell culture condition that induces the mesenchymal-epithelial transition of dedifferentiated porcine retinal pigment epithelial cells

The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of age-related macular degeneration (AMD). Thus, cultured RPE cells are a proper cell model for studying the etiology of AMD in vitro. However, such cultured RPE cells easily undergo epithelial-mesenchymal transition (EMT) that results in changes of cellular morphology and functions of the cells. To restore and maintain the mesenchymal-epithelial transition (MET) of the cultured RPE cells, we cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dishes with DMEM/F12 containing FBS (CC dish-FBS), 2) in petri dishes with DMEM/F12 containing FBS (Petri dish-FBS), 3) in cell culture dishes with DMEM/F12 containing N2 and B27 supplements (CC dish-N2B27), and 4) in petri dishes with DMEM/F12 containing N2 and B27 (Petri dish-N2B27). In addition to observing the cell morphology and behavior, RPE specific markers, as well as EMT-related genes and proteins, were examined by immunostaining, quantitative real-time PCR and Western blotting. The results showed that dedifferentiated pRPE cells maintained EMT in CC dish-FBS, Petri dish-FBS and CC dish-N2B27 groups, whereas MET was induced when the dedifferentiated pRPE cells were cultured in Petri dish-N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE-specific markers and decreased EMT-associated markers. Similar results were observed in induced pluripotent stem cell-derived RPE cells. Furthermore, during the re-differentiation of those dedifferentiated pRPE cells, Petri dish-N2B27 reduced the activity of RhoA and induced F-actin rearrangement, which promoted the nuclear exclusion of transcriptional co-activator with PDZ-binding motif (TAZ) and TAZ target molecule zinc finger E-box binding protein (ZEB1), both of which are EMT inducing factors. This study provides a simple and reliable method to reverse dedifferentiated phenotype of pRPE cells into epithelialized phenotype, which is more appropriate for studying AMD in vitro, and suggests that MET of other cell types might be induced by a similar approach.

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

BackgroundWound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time.MethodsThe 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy.Results The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs.ConclusionsThis acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration

PurposeTransplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area.DesignPhase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832).ParticipantsTwelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults.MethodsSubretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks.Main Outcome MeasuresThe primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT.ResultsFocal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change.ConclusionsSubretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity the impact of cell transplantation and suggest that intervention in early stage of disease should be approached with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection against further deterioration may be evident only after a more extended period of observation.

Smart liposomal drug delivery for treatment of oxidative stress model in human embryonic stem cell-derived retinal pigment epithelial cells

Oxidative stress has been implicated in the progression of age-related macular degeneration (AMD). Treatment with antioxidants seems to delay progression of AMD. In this study, we suggested an antioxidant delivery system based on redox-sensitive liposome composed of phospholipids and a diselenide centered alkyl chain. Dynamic light scattering assessment indicated that the liposomes had an average size of 140 nm with a polydispersity index below 0.2. The percentage of encapsulation efficiency of the liposomes was calculated by high-performance liquid chromatography. The carriers were loaded with N-acetyl cysteine as a model antioxidant drug. We demonstrated responsiveness of the nanocarrier and its efficiency in drug delivery in an oxidative stress model of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The modeled cells treated with diselenide containing liposomes loaded with 10 mM NAC, showed a better therapeutic effect with a cell metabolic activity of 90%, which was significantly higher compared to insensitive liposomes or NAC treated groups (P < 0.05). In addition, the expression of oxidative-sensitive gene markers in diselenide containing liposomes groups were improved. Our results demonstrated fabricated smart liposomes opens new opportunity for targeted treatment of retinal degeneration.

Natural Killer Cell Inhibition by HLA-E Molecules on Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells.

To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can suppress natural killer (NK) cell activation. iPS-RPE cells were cocultured with peripheral blood mononuclear cells (PBMCs) or purified NK cells from healthy donors after stimulation with cytokines. To confirm expression of NK cell-specific markers, flow cytometry and quantitative RT-PCR (qRT-PCR) were performed. NK cells (or PBMCs) cocultured with iPS-RPE cells were assessed for proliferation by Ki-67 expression with flow cytometry, and NK suppression by RPE cells was assessed for granzyme B production with ELISA. Human leukocyte antigen (HLA) expression including HLA-E on iPS-RPE cells was evaluated with flow cytometry and qRT-PCR. The effect of HLA-E downregulation was also investigated using small interfering RNA (siRNA) systems. Following iPS-RPE cell transplantation in vivo, we evaluated NK cell invasion in the retina with immunohistochemistry. Activated NK cells expressed NK-related markers such as CD16, CD56, and CD11b, and NK cells produced cytotoxic agents such as granzyme B, perforin, and TNF-α. Human iPS-RPE cells inhibited cell proliferation and production of these cytotoxic agents by activated NK cells in vitro. iPS-RPE cells constitutively expressed HLA-E and suppressed NK cell activation through an interaction between HLA-E and CD94/NKG2A. Moreover, immunohistochemical evaluation of monkey RPE transplantation into in vivo immune rejection models showed no NK cell invasion in the retina in allografts or xenografts except for one xenografted eye. Cultured iPS cell-derived RPE cells greatly suppress NK cell activation. Thus, NK cells might be inactivated when exposed to this type of retinal cell.

Stem Cell-Based RPE Therapy for Retinal Diseases: Engineering 3D Tissues Amenable for Regenerative Medicine.

Recent clinical trials based on human pluripotent stem cell-derived retinal pigment epithelium cells (hPSC-RPE cells) were clearly a success regarding safety outcomes. However the delivery strategy of a cell suspension, while being a smart implementation of a cell therapy, might not be sufficient to achieve the best results. More complex reconstructed tissue formulations are required, both to improve functionality and to target pathological conditions with altered Bruch's membrane like age-related macular degeneration (AMD). Herein, we describe the various options regarding the stem cell source choices and the different strategies elaborated in the recent years to develop engineered RPE sheets amenable for regenerative therapies.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells.

PurposeAnti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells.MethodsSenescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept.ResultsClinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells.ConclusionsClinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Fibrin hydrogels as a xenofree and rapidly degradable support for transplantation of retinal pigment epithelium monolayers

Stem cell-derived retinal pigment epithelial (RPE) cell transplantation is currently in phase 1 clinical trials for macular degeneration (MD). A major obstacle in these studies is delivering the RPE as a living, flat sheets without leaving behind foreign materials in the retina. Here we investigate the suitability of using hydrogels made from human blood-derived proteins for RPE transplant. Our data shows that these fibrin hydrogels are rigid enough for use in surgery, support growth of stem cell-derived RPE, and are easily degraded within hours without damage to the RPE sheet. These fibrin hydrogels offer a promising solution to transplant RPE for patients with MD.

Collagen vitrigels with low-fibril density enhance human embryonic stem cell-derived retinal pigment epithelial cell maturation.

Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC-RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60nm and Young's modulus of 2.41±0.13MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC-RPE maturation and presents a potential bioengineered substratum for hESC-RPE for future preclinical and clinical applications.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization

BackgroundDysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation.MethodsWe have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions.ResultsUsing highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function.ConclusionsThis report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.