Biomechanical Characterization of Retinal Pigment Epitheliums Derived from hPSCs Using Atomic Force Microscopy.

The retinal pigment epithelium (RPE), a multifunctional cell monolayer located at the back of the eye, plays a crucial role in the survival and homeostasis of photoreceptors. Dysfunction or death of RPE cells leads to retinal degeneration and subsequent vision loss, such as in Age-related macular degeneration and some forms of Retinitis Pigmentosa. Therefore, regenerative medicine that aims to replace RPE cells by new cells obtained from the differentiation of human pluripotent stem cells, is the focus of intensive research. However, despite their critical interest in therapy, there is a lack of biomechanical RPE surface description. Such biomechanical properties are tightly related to their functions. Herein, we used atomic force microscopy (AFM) to analyze both the structural and mechanical properties of RPEs obtained from four cell lines and at different stages of epithelial formation. To characterize epitheliums, we used apical markers in immunofluorescence and showed the increase of transepithelial resistance, as well as the ability to secrete cytokines with an apico-basal polarity. Then, we used AFM to scan the apical surface of living or fixed RPE cells. We show that RPE monolayers underwent softening of apical cell center as well as stiffening of cell borders over epithelial formation. We also observed apical protrusions that depend on actin network, suggesting the formation of microvilli at the surface of RPE epitheliums. These RPE cell characteristics are essential for their functions into the retina and AFM studies may improve the characterization of the RPE epithelium suitable for cell therapy.

Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones.

BACKGROUND: CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such "disease in a dish" models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. METHODS: Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. RESULTS: We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. CONCLUSIONS: This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening.

Photoreceptor Cell Replacement Using Pluripotent Stem Cells: Current Knowledge and Remaining Questions.

Retinal degeneration is an increasing global burden without cure for the majority of patients. Once retinal cells have degenerated, vision is permanently lost. Different strategies have been developed in recent years to prevent retinal degeneration or to restore sight (e.g., gene therapy, cell therapy, and electronic implants). Herein, we present current treatment strategies with a focus on cell therapy for photoreceptor replacement using human pluripotent stem cells. We will describe the state of the art and discuss obstacles and limitations observed in preclinical animal models as well as future directions to improve graft integration and functionality.

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures.

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche.

The Future of Regenerative Medicine: Cell Therapy Using Pluripotent Stem Cells and Acellular Therapies Based on Extracellular Vesicles.

The rapid progress in the field of stem cell research has laid strong foundations for their use in regenerative medicine applications of injured or diseased tissues. Growing evidences indicate that some observed therapeutic outcomes of stem cell-based therapy are due to paracrine effects rather than long-term engraftment and survival of transplanted cells. Given their ability to cross biological barriers and mediate intercellular information transfer of bioactive molecules, extracellular vesicles are being explored as potential cell-free therapeutic agents. In this review, we first discuss the state of the art of regenerative medicine and its current limitations and challenges, with particular attention on pluripotent stem cell-derived products to repair organs like the eye, heart, skeletal muscle and skin. We then focus on emerging beneficial roles of extracellular vesicles to alleviate these pathological conditions and address hurdles and operational issues of this acellular strategy. Finally, we discuss future directions and examine how careful integration of different approaches presented in this review could help to potentiate therapeutic results in preclinical models and their good manufacturing practice (GMP) implementation for future clinical trials.

Corrigendum to "Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration".

[This corrects the article DOI: 10.1155/2019/5637075.].

Human pluripotent stem cells: A toolbox to understand and treat retinal degeneration.

Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP) are retinal degenerative disorders that dramatically damage the retina. As there is no therapeutic option for the majority of patients, vision is progressively and irremediably lost. Owing to their unlimited renewal and potency to give rise to any cell type of the human adult body, human pluripotent stem cells (hPSCs) have been extensively studied in recent years to develop more physiologically relevant in vitro cellular models. Such models open new perspectives to investigate the pathological molecular mechanisms of AMD and RP but also in drug screening. Moreover, proof-of-concept of hPSC-derived retinal cell therapy in animal models have led to first clinical trials. This review outlines the recent advances in the use of hPSCs in pathological modeling of retinal degeneration and their use in regenerative medicine. We also address the associated limitations and challenges that need to be overcome when using hPSCs.

Clinical-grade production and safe delivery of human ESC derived RPE sheets in primates and rodents.

Age-related macular degeneration as well as some forms of Retinitis Pigmentosa (RP) are characterized by a retinal degeneration involving the retinal pigment epithelium (RPE). Various strategies were proposed to cure these disorders including the replacement of RPE cells using human pluripotent stem cells (hPSCs), an unlimited source material to generate in vitro RPE cells. The formulation strategy of the cell therapy (either a reconstructed sheet or a cell suspension) is crucial to achieve an efficient and long lasting therapeutic effect. We previously developed a hPSC-RPE sheet disposed on human amniotic membrane that sustained the vision of rodents with retinal degeneration compared to the same cells injected as a suspension. However, the transplantation strategy was difficult to implement in large animals. Herein we developed two medical devices for the preparation, conservation and implantation of the hPSC-RPE sheet in nonhuman primates. The surgery was safe and well tolerated during the 7-week follow up. The graft integrity was preserved in primates. Moreover, the hPSC-RPE sheet did not induce teratoma or grafted cell dispersion to other organs in rodent models. This work clears the way for the first cell therapy for RP patients carrying RPE gene mutations (LRAT, RPE65 and MERTK).

Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is characterized by retinal pigment epithelial (RPE) cell dysfunction beginning at early stages of the disease. The lack of an appropriate in vitro model is a major limitation in understanding the mechanisms leading to the occurrence of AMD. This study compared human-induced pluripotent stem cell- (hiPSC-) RPE cells derived from atrophic AMD patients (77 y/o ± 7) to hiPSC-RPE cells derived from healthy elderly individuals with no drusen or pigmentary alteration (62.5 y/o ± 17.5). Control and AMD hiPSC-RPE cell lines were characterized by immunofluorescence, flow cytometry, and electronic microscopy. The toxicity level of iron after Fe-NTA treatment was evaluated by an MTT test and by the detection of dichloro-dihydro-fluorescein diacetate. Twelve hiPSC-RPE cell lines (6 AMD and 6 controls) were used for the experiment. Under basal conditions, all hiPSC-RPE cells expressed a phenotypic profile of senescent cells with rounded mitochondria at passage 2. However, the treatment with Fe-NTA induced higher reactive oxygen species production and cell death in hiPSC-RPE AMD cells than in hiPSC-RPE Control cells. Interestingly, functional analysis showed differences in lysosomal activity between the two populations. Indeed, Cathepsin B activity was higher in hiPSC-RPE AMD cells compared to hiPSC-RPE Control cells in basal condition and link to a pH more acidic in this cell population. Moreover, oxidative stress exposure leads to an increase of Cathepsin D immature form levels in both populations, but in a higher proportion in hiPSC-RPE AMD cells. These findings could demonstrate that hiPSC-RPE AMD cells have a typical disease phenotype compared to hiPSC-RPE Control cells.

Developing Cell-Based Therapies for RPE-Associated Degenerative Eye Diseases.

In developed countries, blindness and visual impairment are caused mainly by diseases affecting the retina. These retinal degenerative diseases, including age-related macular dystrophy (AMD) and inherited retinal diseases such as retinitis pigmentosa (RP), are the predominant causes of human blindness worldwide and are responsible for more than 1.5 million cases in France and more than 30 million cases worldwide. Global prevalence and disease burden projections for next 20 years are alarming (Wong et al., Lancet Glob Health 2(2):e106-e116, 2014) and strongly argue toward designing innovative eye-care strategies. At present, despite the scientific advances achieved in the last years, there is no cure for such diseases, making retinal degenerative diseases an unmet medical need.The majority of the inherited retinal disease (IRD) genes codes for proteins acting directly in photoreceptors. Yet, a few of them are expressed in the retinal pigment epithelium (RPE), the supporting tissue necessary for proper functioning of the photoreceptors. Among retinal degenerative diseases, impairment of some RPE genes engenders a spectrum of conditions ranging from stationary visual defects to very severe forms of retinal dystrophies in which the RPE dysfunction leads to photoreceptors cell death and consecutive irreversible vision loss. The accessibility of the eye and the immune privilege of the retina, together with the availability of noninvasive imaging technologies, make such inherited retinal dystrophies a particularly attractive disease model for innovative cell therapy approaches to replace, regenerate, and/or repair the injured RPE tissue. Proof-of-concept studies in animal models have demonstrated the safety and efficacy of the engraftment of therapeutic cells either to support RPE cell functions or to provide a trophic support to photoreceptors. These different approaches are now in the pipeline of drug development with objective to provide first cell-based treatments by 2020.This chapter will focus on the different cell-based strategies developed in the past and current approaches to prevent photoreceptor death in RPE-associated degenerative eye diseases.

Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions.

Dysfunction or death of retinal pigment epithelial (RPE) cells is involved in some forms of Retinitis Pigmentosa and in age-related macular degeneration (AMD). Since there is no cure for most patients affected by these diseases, the transplantation of RPE cells derived from human pluripotent stem cells (hPSCs) represents an attractive therapeutic alternative. First attempts to transplant hPSC-RPE cells in AMD and Stargardt patients demonstrated the safety and suggested the potential efficacy of this strategy. However, it also highlighted the need to upscale the production of the cells to be grafted in order to treat the millions of potential patients. Automated cell culture systems are necessary to change the scale of cell production. In the present study, we developed a protocol amenable for automation that combines in a sequential manner Nicotinamide, Activin A and CHIR99021 to direct the differentiation of hPSCs into RPE cells. This novel differentiation protocol associated with the use of cell culture robots open new possibilities for the production of large batches of hPSC-RPE cells while maintaining a high cell purity and functionality. Such methodology of cell culture automation could therefore be applied to various differentiation processes in order to generate the material suitable for cell therapy.

Cell Therapy for Retinal Dystrophies: From Cell Suspension Formulation to Complex Retinal Tissue Bioengineering.

Retinal degeneration is an irreversible phenomenon caused by various disease conditions including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). During the course of these diseases, photoreceptors (PRs) are susceptible to degeneration due to their malfunctions or to a primary dysfunction of the retinal pigment epithelium (RPE). Once lost, these cells could not be endogenously regenerated in humans, and cell therapy to replace the lost cells is one of the promising strategies to recover vision. Depending on the nature of the primary defect and the stage of the disease, RPE cells, PRs, or both might be transplanted to achieve therapeutic effects. We describe in this review the current knowledge and recent progress to develop such approaches. The different cell sources proposed for cell therapy including human pluripotent stem cells are presented with their advantages and limits. Another critical aspect described herein is the pharmaceutical formulation of the end product to be delivered into the eye of patients. Finally, we also outline the future research directions in order to develop a complex multilayered retinal tissue for end-stage patients.

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch's membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

hRPE cells derived from induced pluripotent stem cells are more sensitive to oxidative stress than ARPE-19 cells.

The ARPE-19 cell line is currently used as an in vitro model for retinal diseases such as age-related degeneration (AMD). However, several studies have pointed out morphological and genetic differences between ARPE-19 cells and human fetal or adult retinal pigment epithelial (hRPE) cells. This study aims to compare ARPE-19 cells to hRPE cells derived from human induced pluripotent stem cells (hiPSCs) in both normal and oxidative stress conditions induced by Fe-NTA treatment. Indeed, oxidative stress is an essential contributing factor in AMD. hiPSC obtained from peripheral venous blood samples or fibroblasts of individuals aged over 60 years were first reprogrammed to hiPSC and then differentiated into RPE cells. In contrast to ARPE-19 cells, hiPSC-RPE cells expressed ß-galactosidase activity, suggesting that only the latter display signs of senescence. Treatment with 10 mM of FeNTA induced a higher reactive oxygen species (ROS) production and increased cell death in hiPSC-RPE cells compared to ARPE-19 cells. Moreover, morphological analysis and Annexin V and Propidium iodide (PI) test suggested a necrotic cell death pattern induced by treatment in hiPSC-RPE cells that is not observed in ARPE-19 cells. Taken as a whole, our findings suggest that hiPSC-RPE cells are more sensitive to oxidative stress than ARPE-19 cells.

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.

Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration.

Replacing defective retinal pigment epithelial (RPE) cells with those derived from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) is a potential strategy for treating retinal degenerative diseases. Early clinical trials have demonstrated that hESC-derived or hiPSC-derived RPE cells can be delivered safely as a suspension to the human eye. The next step is transplantation of hESC/hiPSC-derived RPE cells as cell sheets that are more physiological. We have developed a tissue-engineered product consisting of hESC-derived RPE cells grown as sheets on human amniotic membrane as a biocompatible substrate. We established a surgical approach to engraft this tissue-engineered product into the subretinal space of the eyes of rats with photoreceptor cell loss. We show that transplantation of the hESC-RPE cell sheets grown on a human amniotic membrane scaffold resulted in rescue of photoreceptor cell death and improved visual acuity in rats with retinal degeneration compared to hESC-RPE cells injected as a cell suspension. These results suggest that tissue-engineered hESC-RPE cell sheets produced under good manufacturing practice conditions may be a useful approach for treating diseases of retinal degeneration.

Use of human pluripotent stem cells to study and treat retinopathies.

Human cell types affected by retinal diseases (such as age-related macular degeneration or retinitis pimentosa) are limited in cell number and of reduced accessibility. As a consequence, their isolation for in vitro studies of disease mechanisms or for drug screening efforts is fastidious. Human pluripotent stem cells (hPSCs), either of embryonic origin or through reprogramming of adult somatic cells, represent a new promising way to generate models of human retinopathies, explore the physiopathological mechanisms and develop novel therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be used to study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, can be genetically converted to induce pluripotent stem cells together with the continuous improvement of methods to differentiate these cells into disease-affected cellular subtypes opens new perspectives to model and understand a large number of human pathologies, including retinopathies. This review focuses on the added value of hPSCs for the disease modeling of human retinopathies and the study of their molecular pathological mechanisms. We also discuss the recent use of these cells for establishing the validation studies for therapeutic intervention and for the screening of large compound libraries to identify candidate drugs.

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

Human induced pluripotent stem cells as a tool to model a form of Leber congenital amaurosis.

Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA) and to model this type of LCA for drug screening. Fibroblasts from two unrelated clinically identified patients with a yet undetermined gene mutation were reprogrammed to pluripotency by retroviral transduction. These human induced pluripotent stem cells (hiPSCs) were differentiated into neural stem cells (NSCs) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome-wide transcriptome analysis was performed with Affymetrix Exon Array GeneChip(®), comparing LCA-hiPSCs derivatives to controls. A genomic search for alteration in all genes known to be involved in LCA revealed a common polymorphism on the GUCY2D gene, referenced as the LCA type I (OMIM *600179 and #204000), but the causative gene remained unknown. The hiPSCs expressed the key pluripotency factors and formed embryoid bodies in vitro containing cells originating from all three germ layers. They were successfully differentiated into NSC and RPE cells. One gene, NNAT, was upregulated in LCA cell populations, and three genes were downregulated, GSTT1, TRIM61 and ZNF558, with potential correlates for molecular mechanisms of this type of LCA, in particular for protein degradation and oxidative stress. The two LCA patient-specific iPSC lines will contribute to modeling LCA phenotypes and screening candidate drugs.