Stem Cell-derived Retinal Pigment Epithelium Research Articles

The Fate of RPE Cells Following hESC-RPE Patch Transplantation in Haemorrhagic Wet AMD: Pigmentation, Extension of Pigmentation, Thickness of Transplant, Assessment for Proliferation and Visual Function-A 5 Year-Follow Up.

(1) Background: We reviewed a stem cell-derived therapeutic strategy for advanced neovascular age-related macular degeneration (nAMD) using a human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) monolayer delivered on a coated, synthetic basement membrane (BM)-the patch-and assessed the presence and distribution of hESC-RPE over 5 years following transplantation, as well as functional outcomes. (2) Methods: Two subjects with acute vision loss due to sub-macular haemorrhage in advanced nAMD received the hESC-RPE patch. Systematic immunosuppression was used peri-operatively followed by local depot immunosuppression. The subjects were monitored for five years with observation of RPE patch pigmentation, extension beyond the patch boundary into surrounding retina, thickness of hESC-RPE and synthetic BM and review for migration and proliferation of hESC-RPE. Visual function was also assessed. (3) Results: The two study participants showed clear RPE characteristics of the patch, preservation of some retinal ultrastructure with signs of remodelling, fibrosis and thinning on optical coherence tomography over the 5-year period. For both participants, there was evidence of pigment extension beyond the patch continuing until 12 months post-operatively, which stabilised and was preserved until 5 years post-operatively. Measurement of hESC-RPE and BM thickness over time for both cases were consistent with predefined histological measurements of these two layers. There was no evidence of distant RPE migration or proliferation in either case beyond the monolayer. Sustained visual acuity improvement was apparent for 2 years in both subjects, with one subject maintaining the improvement for 5 years. Both subjects demonstrated initial improvement in fixation and microperimetry compared to baseline, at year 1, although only one maintained this at 4 years post-intervention. (4) Conclusions: hESC-RPE patches show evidence of continued pigmentation, with extension, to cover bare host basement membrane for up to 5 years post-implantation. There is evidence that this represents functional RPE on the patch and at the patch border where host RPE is absent. The measurements for thickness of hESC-RPE and BM suggest persistence of both layers at 5 years. No safety concerns were raised for the hypothetical risk of RPE migration, proliferation or tumour formation. Visual function also showed sustained improvement for 2 years in one subject and 5 years in the other subject.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

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.

Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid β-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells

Background aimsAge-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. MethodsReplacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. ResultsWe found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2–5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium–derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. ConclusionsThe possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation

IntroductionThe retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. MethodsA retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. ResultsGelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. ConclusionGelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.

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

PurposeTo report long-term results from a Phase 1/2a clinical trial assessment of a scaffold-based human embryonic stem cell-derived retinal pigmented epithelium (RPE) implant in subjects with advanced geographic atrophy (GA). DesignThe study was a US Food and Drug Administration (FDA)-approved single-arm, open-label Phase 1/2a clinical trial. SubjectsSubjects were between the ages of 69 and 85 at the time of enrollment and were legally blind in the treated eye (best corrected visual acuity (BCVA) of ≤ 20/200) as a result of GA involving the fovea. InterventionThe clinical trial enrolled 16 subjects, 15 of whom were successfully implanted. The implant was administered to the worse-seeing eye with the use of a custom subretinal insertion device. The companion non-implanted eye served as the control. The primary endpoint was at 1 year; thereafter subjects were followed at least yearly. Main Outcome MeasuresSafety was the primary endpoint of the study. The occurrence and frequency of adverse events were determined by scheduled eye exams, including measurement of BCVA, intraocular pressure, and multimodal imaging (fundus photography, optical coherence tomography, and fluorescein angiography). In addition, serum antibody titers were collected to monitor systemic humoral immune responses to the implant cells. ResultsAt a median follow-up of three years, fundus photography revealed no migration of the implant. There were no unanticipated, severe, implant-related adverse events throughout the follow-up period, and the most common anticipated severe adverse event (severe retinal hemorrhage) was eliminated in the second cohort (9 subjects) through the use of improved intraoperative hemostasis. Non-severe, transient retinal hemorrhages were noted either intra-op or post-op in all subjects as anticipated for a subretinal surgical procedure. Throughout the median 3-year follow-up, the results show that implanted eyes were more likely to improve by >5 letters BCVA, and less likely to decrease by >5 letters compared to non-implanted eyes. ConclusionsThis report details the long-term follow-up of the largest study of subjects with GA to receive a scaffold-based stem cell-derived bioengineered RPE implant. Results show that the implant, at a median 3-year follow-up, is safe and well-tolerated in subjects with advanced dry AMD. The safety profile, along with the early indication of efficacy, warrants further clinical evaluation of this novel approach for the treatment of GA.

Micromolded honeycomb scaffold design to support the generation of a bilayered RPE and photoreceptor cell construct.

Age-related macular degeneration (AMD) causes blindness due to loss of retinal pigment epithelium (RPE) and photoreceptors (PRs), which comprise the two outermost layers of the retina. Given the small size of the macula and the importance of direct contact between RPE and PRs, the use of scaffolds for targeted reconstruction of the outer retina in later stage AMD and other macular dystrophies is particularly attractive. We developed microfabricated, honeycomb-patterned, biodegradable poly(glycerol sebacate) (PGS) scaffolds to deliver organized, adjacent layers of RPE and PRs to the subretinal space. Furthermore, an optimized process was developed to photocure PGS, shortening scaffold production time from days to minutes. The resulting scaffolds robustly supported the seeding of human pluripotent stem cell-derived RPE and PRs, either separately or as a dual cell-layered construct. These advanced, economical, and versatile scaffolds can accelerate retinal cell transplantation efforts and benefit patients with AMD and other retinal degenerative diseases.

Medium Depth Influences O2 Availability and Metabolism in Human RPE Cultures.

Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. We altered the availability of O2 to human primary and induced pluripotent stem cell-derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, 13C6-glucose and 13C5-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation. Medium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood." As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.

A biologically validated mathematical model for decoding epithelial apical, basolateral, and paracellular electrical properties.

Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances (n = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology.NEW & NOTEWORTHY This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.

Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy.

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.

Updates on Emerging Interventions for Autosomal Recessive ABCA4-Associated Stargardt Disease.

Autosomal recessive Stargardt disease (STGD1) is an inherited retinal degenerative disease associated with a mutated ATP-binding cassette, subfamily A, member 4 (ABCA4) gene. STGD1 is the most common form of juvenile macular degeneration with onset in late childhood to early or middle adulthood and causes progressive, irreversible visual impairment and blindness. No effective treatment is currently available. In the present article, we review the most recent updates in clinical trials targeting the management of STGD1, including gene therapy, small molecule therapy, and stem cell therapy. In gene therapy, dual adeno-associated virus and non-viral vectors have been successful in delivering the human ABCA4 gene in preclinical studies. For pharmaceutical therapies ALK-001, deuterated vitamin A shows promise with preliminary data for phase 2 trial, demonstrating a decreased atrophy growth rate after two years. Stem cell therapy using human pluripotent stem cell-derived retinal pigment epithelium cells demonstrated long-term safety three years after implantation and visual acuity improvements in the first two years after initiation of therapy. Many other treatment options have ongoing investigations and clinical trials. While multiple potential interventions have shown promise in attenuating disease progression, further exploration is necessary to demonstrate treatment safety and efficacy.

Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells.

To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover. Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting. Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression. Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy.

A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells

Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Considerations for Developing an Autologous Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) Replacement Therapy.

Cell-replacement therapies are a new class of treatments, which include induced pluripotent stem cell (iPSC)-derived tissues that aim to replace degenerated cells. iPSCs can potentially be used to generate any cell type of the body, making them a powerful tool for treating degenerative diseases. Cell replacement for retinal degenerative diseases is at the forefront of cell therapies, given the accessibility of the eye for surgical procedures and a huge unmet medical need for retinal degenerative diseases with no current treatment options. Clinical trials are ongoing in different parts of the world using stem cell-derived retinal pigment epithelium (RPE). This review focuses on scientific and regulatory considerations when developing an iPSC-derived RPE cell therapy from the development of a robust and efficient differentiation protocol to critical quality control assays for cell validation, the choice of an appropriate animal model for preclinical testing, and the regulatory aspects that dictate the final approval for proceeding to a first-in-human clinical trial.

Progress In Stem Cells Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery.

Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE derivation, their delivery, and recent clinical studies related to pRPE transplantation in patients. We have also discussed a few limitations with in vitro RPE culture and potential solutions to overcome such problems which can be helpful in developing functional RPE tissue.

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Single-cell transcriptomics reveals maturation of transplanted stem cell–derived retinal pigment epithelial cells toward native state

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched invitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all invitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.