Retinal Pigment Epithelium Markers Research Articles

Influence of Substrate Stiffness on iPSC-Derived Retinal Pigmented Epithelial Cells.

Retinal degenerative diseases are a major cause of blindness involving the dysfunction of photoreceptors, retinal pigmented epithelium (RPE), or both. A promising treatment approach involves replacing these cells via surgical transplantation, and previous work has shown that cell delivery scaffolds are vital to ensure sufficient cell survival. Thus, identifying scaffold properties that are conducive to cell viability and maturation (such as suitable material and mechanical properties) is critical to ensuring a successful treatment approach. In this study, we investigated the effect of scaffold stiffness on human RPE attachment, survival, and differentiation, comparing immortalized (ARPE-19) and stem cell-derived RPE (iRPE) cells. Polydimethylsiloxane was used as a model polymer substrate, and varying stiffness (~12 to 800 kPa) was achieved by modulating the cross-link-to-base ratio. Post-attachment changes in gene and protein expression were assessed using qPCR and immunocytochemistry. We found that while ARPE-19 and iRPE exhibited significant differences in morphology and expression of RPE markers, substrate stiffness did not have a substantial impact on cell growth or maturation for either cell type. These results highlight the differences in expression between immortalized and iPSC-derived RPE cells, and also suggest that stiffnesses in this range (~12-800 kPa) may not result in significant differences in RPE growth and maturation, an important consideration in scaffold design.

Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction

ObjectiveStem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction.MethodsRPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining.ResultsThe rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF.ConclusionRPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Nanofibrous scaffolds with ex vivo cultured human iPSC‐derived RPE cells for transplantation into porcine eyes

AbstractPurpose: There are several retinal degenerative diseases including age‐related macular degeneration (AMD) where the retinal pigment epithelium (RPE) is affected primarily and photoreceptors secondarily. The RPE replacement based on changing of diseased RPE by ex vivo cultured iPSC‐derived RPE cells on nanofibrous membranes can prolong the survival of adjacent photoreceptors.Methods: The feeder‐free system of culturing and differentiation of human iPSCs was used. The phenotypic maturation was examined using immunolabelling for the differentiation and polarization markers. Scaffolds with confluent human iPSC‐derived RPE were implanted into the right eye of 7 minipigs using 3‐port pars plana vitrectomy. Because the human cells were transplanted into the porcine eye (xenogeneic transplantation), immunosuppression therapy with Tacrolimus was applied. At 1–4 weeks upon transplantation, noninvasive examinations of the implanted retina were performed by OCT and fundus camera.Results: The iPSC‐derived RPE cells showed typical cellular morphology and express the RPE markers such as ZO1, BEST, RPE65, and others, with no traces of pluripotent stem cells. Scaffolds with seeded RPE were successfully implanted into subretinal space of porcine eyes. OCT and fundus camera examinations were able to localize the implant in the retina and demonstrated a normal neuroretina in the place of the implant with healed retinotomy, without inflammatory reaction on the implant.Conclusions: In conclusion, we can state that the functional and morphological properties of human iPSC‐derived RPE cells cultured on nanomembranes confirmed the good quality of this epithelium including proper differentiation, confluency of the polarized monolayer, and ability to long‐term survival. The correct placing of implants in the subretinal space testified to the rightness of employed surgical techniques and functionality of the delivery instrumentarium.

Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient.

We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE) cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide potential cell sources for both basic scientific research and clinical application. In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR), karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and flow cytometry. The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated 99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC- RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1, Bestrophin, and PMEL17). A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with native RPE characteristics, which might provide potential regenerative treatments for AMD patients.

Accelerated maturation of ARPE‐19 cells for the translational assessment of gene therapy

AbstractPurpose: Retinal pigment epithelium (RPE) modulates the exchange of metabolite products to and from the retina, and are paramount to maintain retinal function. The human RPE cell‐line ARPE‐19 is commercially available and widely used as an alternative to primary RPE. However, these cells undergo epithelial‐mesenchymal transition (EMT) losing many features of primary RPE such as polarity, pigmentation, and expression of RPE signature markers. We aimed to determine whether a combination of RPE‐specific laminin (LN) coating and media supplementation with nicotinamide (NAM) could improve ARPE‐19 expression of genes and proteins, along with morphological phenotype resembling mature RPE to improve assessment of RPE‐specific gene therapy strategies.Methods: ARPE‐19 cells were propagated on tissue culture plastic with or without NAM supplementation and/or coated with human recombinant LN‐521. Cell morphology was evaluated by phase microscopy. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno associated virus (AAV)1 or AAV2, VMD2‐driven GFP, were assessed at 2‐ and 4‐weeks post‐plating in the different culturing condition with low multiplicity of infection.Results: The combination of LN‐521 coating with NAM supplementation promoted cytoskeletal and tight junction proteins reorganization, and expression of differentiation markers such as VMD2, RPE65 and PDGFRB. In addition, the combination reduced the expression of the EMT markers, TNFα and TGF‐β1; and NCAM1, an immature phenotype RPE marker. Both AAV1‐ and AAV2‐VMD2‐GFP expression was detected from 2‐weeks and improved at 4‐weeks post‐plating.Conclusions: Our study demonstrates that the combination of NAM with LN‐521 accelerated the kinetics of expression of mature RPE signature genes, reduced expression of EMT genes, accompanied with improved epithelial cell morphology and cytoskeletal organization, bringing the dedifferentiated ARPE‐19 closer to their in vivo phenotype. Moreover, viral‐mediated RPE‐specific gene expression demonstrates the amenability of the combinatorial cultures for translational assessment of RPE gene therapies.

Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium.

The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program.

Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture

ObjectiveRecent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to...

Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE–derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting “immature” gene regulatory networks.

Intraretinal pigmented cells in retinal degenerative disease

PurposeInvasion of pigmented cells into the retina occurs in retinal degenerative diseases, such as macular telangiectasia type 2 (MacTel) and retinitis pigmentosa (RP). These intraretinal pigmented cells may be derived...

Development-related mitochondrial properties of retinal pigment epithelium cells derived from hEROs.

To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy (TEM) was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and evaluated by flow cytometry, reactive oxygen species (ROS) levels were evaluated by flow cytometry, and adenosine triphosphate (ATP) levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed. hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers (Pax6, MITF, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hEROs-RPE and SD-RPE cells. The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80. However, the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80, with hEROs-RPE mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria. From the mitochondrial perspective, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.

LncRNA MEG3, Acting as a ceRNA, Modulates RPE Differentiation Through the miR-7-5p/Pax6 Axis.

Accumulated evidence indicated that long non-coding RNAs (lncRNAs) involves in numerous biological and pathological processes, including age-related macular degeneration (AMD). Dysfunction and dedifferentiation of retinal pigment epithelium (RPE) cells have been demonstrated to be one of the crucial factor in AMD etiology. Herein, we aim to investigate the essential role of lncRNA maternally expressed gene 3 (MEG3) in AMD progression. Expression patterns of MEG3 were measured in dysfunctional REP cells exposed with H2O2 or TNF-α using qRT-PCR assay. Specifically, the intercellular distribution of MEG3 in REP cells was further explored using the subcellular fraction detection. Relative expression of RPE markers or RPE dedifferentiation-related markers was determined using qRT-PCR and western blot analysis, respectively. Immunofluorescence staining was performed to examine the expressions of RPE markers ZO-1 and β-catenin. Concentration of vascular endothelial growth factor (VEGFA) in the supernatant was detected using ELISA kit. Luciferase reporter assay was performed to verify the MEG3/miR-7-5p/Pax6 regulatory network, which was further determined in in vitro studies. MEG3 expression was significantly decreased in H2O2 or TNF-α-treated REP cells, and it was upregulated along with RPE differentiation. Reduced MEG3 expression resulted in RPE dedifferentiation, which was indicated by decreased expressions of RPE markers, accumulated mitochondrial reactive oxygen species, and reduced VEGFA. Mechanistically, MEG3 functioned as a sponge for miR-7-5p to restore the expression of Pax6. Our study demonstrated that MEG3 exerts a protective role against AMD by maintaining RPE differentiation via miR-7-5p/Pax6 axis, suggesting a protective therapeutic target in AMD treatment.

Efficient differentiation of human embryonic stem cells to retinal pigment epithelium under defined conditions

Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision.ObjectiveThe objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use.DesignA protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function.ResultsWe show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE.ConclusionThis study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

SummaryRecent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

Немедикаментозные методы лечения поздней стадии возрастной макулярной дегенерации

Age-related macular degeneration (AMD) is the leading cause of blindness in people over 55 in developed countries. Moreover, the number of these patients will increase growth as life expectancy increases. It is estimated that late AMD accounts for half of blindness and low vision cases in European countries. A myriad of studies is currently underway to discover cutting-edge, effective therapeutic modalities. Gene therapy is a novel alternative to regular intravitreal injections of anti-VEGF agents for late wet AMD. This technique’s heart is a specific gene delivery to target cells to generate natural VEGF inhibitors. Gene therapy affecting the complement system to deactivate its end product, the membrane attack complex, is reasonable in late atrophic AMD. Studies on stem cell therapy for late atrophic AMD undergo as well. It was demonstrated that retinal pigment epithelium (RPE) cells derived from human embryonic stem cells or induced pluripotent stem cells express typical RPE markers that can phagocytize photoreceptor segments. Electrical stimulation and magnet therapy are already introduced into clinical practice to rehabilitate patients with late AMD. Magnetic and electrical fields improve impulse transmitting, activate intracellular and tissue regeneration of the retina. Recent findings are promising but require further in-depth studies. Keywords: age-related macular degeneration, retinal scar, gene therapy, stem cells, physiotherapy, rehabilitative medicine. For citation: Drakon A.K., Kurguzova A.G., Sheludchenko V.M., Korchazhkina N.B. Non-medical treatment for late age-related macular degeneration. Russian Journal of Clinical Ophthalmology. 2021;21(4):215–219 (in Russ.). DOI: 10.32364/2311-7729-2021-21-4-215-219.

Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

Sphere-induced reprogramming of RPE cells into dual-potential RPE stem-like cells.

BackgroundThe retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols.MethodsSphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches.FindingsWe provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming.InterpretationThe Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs.FundNational Natural Science Foundation of China and the National Institute of Health USA.

LINC00167 Regulates RPE Differentiation by Targeting the miR-203a-3p/SOCS3 Axis

Increasing evidence has indicated that long non-coding RNAs (lncRNAs) play significant roles in various diseases; however, their roles in age-related macular degeneration (AMD) remain unclear. Dedifferentiation and dysfunction of retinal pigment epithelium (RPE) cells have been shown to contribute to AMD etiology in several studies. Herein, we found that lncRNA LINC00167 was downregulated in RPE-choroid samples of AMD patients and dysfunctional RPE cells, and it was consistently upregulated along with RPE differentiation. In vitro study indicated that reduced endogenous LINC00167 expression resulted in RPE dedifferentiation, which was typified by attenuated expression of RPE markers, reduced vascular endothelial growth factor A secretion, accumulation of mitochondrial reactive oxygen species, and interrupted phagocytic ability. Mechanistically, LINC00167 functioned as a sponge for microRNA miR-203a-3p to restore the expression of the suppressor of cytokine signaling 3 (SOCS3), which further inhibited the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Taken together, our study demonstrated that LINC00167 showed a protective role in AMD by maintaining RPE differentiation through the LINC00167/miR-203a-3p/SOCS3 axis and might be a potential therapeutic target for AMD.

APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy. Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed. High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF. Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.