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.

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.

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).

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.

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.

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.

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.

Organotypic heart slices for cell transplantation and physiological studies.

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drugs for studying electrophysiology and developing cellular therapeutic approaches to alleviate cardiac tissue damage.

An in vitro beating heart model for long-term assessment of experimental therapeutics.

AIMS: Within the framework of studies aiming at regenerative medicine for cardiovascular disease, we have developed an in vitro model to analyse human embryonic stem (ES) cell engraftment into the myocardium. METHODS AND RESULTS: This model is based on organotypic rat ventricular slices maintained in culture at the air-medium interface on semi-porous membranes. Survival and differentiation of human cardiomyocytes derived from ES cells were then assessed for several months. In addition, we observed that ventricular tissue slices not only exhibited normal histology, but also rhythmic contractions till the end of the experiments (up to 3 months). Similar results were obtained using ventricular slices obtained from two human foetuses at 8 and 9.5 weeks of age. Calcium transients were associated with the beating frequency, and the pattern was modulated in a dose-dependent manner by epinephrine. CONCLUSION: Our data suggest that the organotypic heart slice culture on semi-porous membranes is a relevant in vitro heart model for long-term histological and physiological studies.

Interferon-alpha gene therapy by lentiviral vectors contrasts ovarian cancer growth through angiogenesis inhibition.

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Recruitment of human umbilical vein endothelial cells and human primary fibroblasts into experimental tumors growing in SCID mice.

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.

Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors.

Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy approaches to ovarian cancer aimed at achieving long-term expression of therapeutic genes.

Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability.

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene. These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.