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.

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.

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.