Publications by authors named "Olivier Goureau"

47 Publications

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy.

Front Cell Neurosci 2021 18;15:648210. Epub 2021 Mar 18.

Institut de la Vision, Sorbonne Université, Paris, France.

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.
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http://dx.doi.org/10.3389/fncel.2021.648210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012682PMC
March 2021

Reproducing diabetic retinopathy features using newly developed human induced-pluripotent stem cell-derived retinal Müller glial cells.

Glia 2021 Mar 8. Epub 2021 Mar 8.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC-associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post-mortem retinal tissues, the donor scarcity limits their use. To overcome this problem, we developed a protocol to generate and bank human induced pluripotent stem cell-derived MGCs (hiMGCs). Using a transcriptome analysis, we showed that the three genetically independent hiMGCs generated were homogeneous and showed phenotypic characteristics and transcriptomic profile of pMGCs. These cells expressed key MGC markers, including Vimentin, CLU, DKK3, SOX9, SOX2, S100A16, ITGB1, and CD44 and could be cultured up to passage 8. Under our culture conditions, hiMGCs and pMGCs expressed low transcript levels of RLPB1, AQP4, KCNJ1, KCJN10, and SLC1A3. Using a disease modeling approach, we showed that hiMGCs could be used to model the features of diabetic retinopathy (DR)-associated dyslipidemia. Indeed, palmitate, a major free fatty acid with elevated plasma levels in diabetic patients, induced the expression of inflammatory cytokines found in the ocular fluid of DR patients such as CXCL8 (IL-8) and ANGPTL4. Moreover, the analysis of palmitate-treated hiMGC secretome showed an upregulation of proangiogenic factors strongly related to DR, including ANG2, Endoglin, IL-1β, CXCL8, MMP-9, PDGF-AA, and VEGF. Thus, hiMGCs could be an alternative to pMGCs and an extremely valuable tool to help to understand and model glial cell involvement in retinal disorders, including DR.
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http://dx.doi.org/10.1002/glia.23983DOI Listing
March 2021

A Splice Variant in Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells.

Cells 2021 Jan 18;10(1). Epub 2021 Jan 18.

Institut de la Vision, CNRS, INSERM, Sorbonne Université, 17 rue Moreau, F-75012 Paris, France.

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.
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http://dx.doi.org/10.3390/cells10010179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831140PMC
January 2021

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Front Cell Dev Biol 2020 27;8:585675. Epub 2020 Oct 27.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.
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http://dx.doi.org/10.3389/fcell.2020.585675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652757PMC
October 2020

SARS-CoV-2 targets neurons of 3D human brain organoids.

EMBO J 2020 10 23;39(20):e106230. Epub 2020 Sep 23.

Institute of Human Genetics, University Hospital Düsseldorf, Heinrich-Heine-Universität, Düsseldorf, Germany.

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.
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http://dx.doi.org/10.15252/embj.2020106230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560208PMC
October 2020

Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids.

Light Sci Appl 2020 17;9:140. Epub 2020 Aug 17.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, F-75012 Paris, France.

Optical coherence tomography offers astounding opportunities to image the complex structure of living tissue but lacks functional information. We present dynamic full-field optical coherence tomography as a technique to noninvasively image living human induced pluripotent stem cell-derived retinal organoids. Coloured images with an endogenous contrast linked to organelle motility are generated, with submicrometre spatial resolution and millisecond temporal resolution, creating a way to identify specific cell types in living tissue via their function.
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http://dx.doi.org/10.1038/s41377-020-00375-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429964PMC
August 2020

[Retinal organoids as a new tool for understanding and treating retinal diseases].

Med Sci (Paris) 2020 Jun-Jul;36(6-7):626-632. Epub 2020 Jul 2.

Institut de la Vision, Sorbonne Université, Inserm, CNRS, 17 rue Moreau, F-75012 Paris, France.

Generation of retinal organoids from pluripotent stem cells represents an important advance in the study of retinal development and offer new perspectives for the study of retinal diseases missing suitable animal models. Understanding the key stages of retinal development in vertebrates enabled to design protocols to generate self-organized three-dimensional structures derived from pluripotent stem cells and containing all retinal cell types. In addition to their application in basic research, such as the characterization of molecular and cellular mechanisms in retinal pathophysiology, these miniature organs also open up encouraging prospects in the field of cell therapy or the screening of therapeutic molecules, although some obstacles remain to be overcome.
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http://dx.doi.org/10.1051/medsci/2020098DOI Listing
October 2020

[Photoreceptor cell transplantation for future treatment of retinitis pigmentosa].

Med Sci (Paris) 2020 Jun-Jul;36(6-7):600-606. Epub 2020 Jul 2.

Institut de la Vision, Sorbonne Université, Inserm, CNRS, 17 rue Moreau, F-75012 Paris, France.

In inherited retinal diseases such retinitis pigmentosa, characterized by progressive loss of light sensitive neurons (photoreceptors), cell therapy is now considered as an attractive strategy. Photoreceptor cell replacement would be valuable for restoring function to retinas in a way that is independent from the cause of the disease. With advances in stem cell biology, considerable strides have been made towards the generation of retinal cells, in particular with the development of 3D culture systems allowing the generation of retinal organoids from pluripotent stem cells. In this review, we present a state-of-the art of preclinical strategies conducted in animal models for photoreceptor replacement from stem cell-derived photoreceptors and we discuss the important obstacles to overcome in the future.
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http://dx.doi.org/10.1051/medsci/2020097DOI Listing
October 2020

AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Int J Mol Sci 2020 Feb 3;21(3). Epub 2020 Feb 3.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, 75012 Paris, France.

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.
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http://dx.doi.org/10.3390/ijms21030994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036814PMC
February 2020

The primate model for understanding and restoring vision.

Proc Natl Acad Sci U S A 2019 Dec 23. Epub 2019 Dec 23.

Institut de la Vision, INSERM, CNRS, Sorbonne Université, F-75012 Paris, France;

Retinal degenerative diseases caused by photoreceptor cell death are major causes of irreversible vision loss. As only primates have a macula, the nonhuman primate (NHP) models have a crucial role not only in revealing biological mechanisms underlying high-acuity vision but also in the development of therapies. Successful translation of basic research findings into clinical trials and, moreover, approval of the first therapies for blinding inherited and age-related retinal dystrophies has been reported in recent years. This article explores the value of the NHP models in understanding human vision and reviews their contribution to the development of innovative therapeutic strategies to save and restore vision.
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http://dx.doi.org/10.1073/pnas.1902292116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936588PMC
December 2019

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

Biomaterials 2020 02 6;230:119603. Epub 2019 Nov 6.

INSERM U861, I-Stem, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 91100, Corbeil-Essonnes, France; UEVE U861, I-Stem, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 91100, Corbeil-Essonnes, France. Electronic address:

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).
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http://dx.doi.org/10.1016/j.biomaterials.2019.119603DOI Listing
February 2020

Generation of human induced pluripotent stem cell lines from a patient with ITM2B-related retinal dystrophy and a non mutated brother.

Stem Cell Res 2019 12 5;41:101625. Epub 2019 Nov 5.

INSERM, CNRS, Institut de la Vision, Sorbonne Université, 17 rue Moreau, Paris, F-75012, France; CHNO des Quinze-Vingts, INSERM-DGOS CIC 1423, 28 rue de Charenton, Paris, F-75012, France. Electronic address:

Human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of a patient affected with an autosomal dominant retinal dystrophy carrying the mutation c.782A>C, p.Glu261Ala in ITM2B and from an unaffected brother. Three different iPSC lines were generated and characterized from primary dermal fibroblasts of the affected subject and two from the unaffected brother. All iPSC lines expressed the pluripotency markers, were able to differentiate into the three germ layers and presented normal karyotypes. This cellular model will provide a powerful tool to study this retinal dystrophy and better understand the role of ITM2B.
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http://dx.doi.org/10.1016/j.scr.2019.101625DOI Listing
December 2019

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

Nat Commun 2019 10 4;10(1):4524. Epub 2019 Oct 4.

Sorbonne Université, Institut de la Vision, INSERM, CNRS, 75012, Paris, France.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.
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http://dx.doi.org/10.1038/s41467-019-12330-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778196PMC
October 2019

Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells.

Stem Cells Int 2019 15;2019:7858796. Epub 2019 Jul 15.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 Paris, France.

The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases.
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http://dx.doi.org/10.1155/2019/7858796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664555PMC
July 2019

Photoreceptor cell replacement in macular degeneration and retinitis pigmentosa: A pluripotent stem cell-based approach.

Prog Retin Eye Res 2019 07 16;71:1-25. Epub 2019 Mar 16.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, F-75012, Paris, France. Electronic address:

The human retina fails to regenerate and cell-based therapies offer options for treatment of blinding retinal diseases, such as macular degeneration and retinitis pigmentosa. The last decade has witnessed remarkable advances in generation of retinal cells and retinal tissue from human pluripotent stem cells (PSCs). The development of 3D culture systems allowing generation of human retinal organoids has substantially increased the access to human material for future clinical applications aiming at replacing the lost photoreceptors in retinal degenerative diseases. This review outlines the advances that have been made toward generation and characterization of transplantable photoreceptors from PSCs and summarizes the current status of PSC-based preclinical studies for photoreceptor replacement conducted in animal models. Considering the recent turning point in our understanding of donor photoreceptor integration, this review focuses on the most crucial obstacles that hinder the photoreceptor replacement, discusses the most promising strategies to overcome them in the future, and provides a perspective on the approaching advancement in the application of PSC technology for treatment of photoreceptor degenerative diseases.
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http://dx.doi.org/10.1016/j.preteyeres.2019.03.001DOI Listing
July 2019

Optogenetic Light Sensors in Human Retinal Organoids.

Front Neurosci 2018 2;12:789. Epub 2018 Nov 2.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.
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http://dx.doi.org/10.3389/fnins.2018.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224345PMC
November 2018

Pluripotent Stem Cell-Based Approaches to Explore and Treat Optic Neuropathies.

Front Neurosci 2018 20;12:651. Epub 2018 Sep 20.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

Sight is a major sense for human and visual impairment profoundly affects quality of life, especially retinal degenerative diseases which are the leading cause of irreversible blindness worldwide. As for other neurodegenerative disorders, almost all retinal dystrophies are characterized by the specific loss of one or two cell types, such as retinal ganglion cells, photoreceptor cells, or retinal pigmented epithelial cells. This feature is a critical point when dealing with cell replacement strategies considering that the preservation of other cell types and retinal circuitry is a prerequisite. Retinal ganglion cells are particularly vulnerable to degenerative process and glaucoma, the most common optic neuropathy, is a frequent retinal dystrophy. Cell replacement has been proposed as a potential approach to take on the challenge of visual restoration, but its application to optic neuropathies is particularly challenging. Many obstacles need to be overcome before any clinical application. Beyond their survival and differentiation, engrafted cells have to reconnect with both upstream synaptic retinal cell partners and specific targets in the brain. To date, reconnection of retinal ganglion cells with distal central targets appears unrealistic since central nervous system is refractory to regenerative processes. Significant progress on the understanding of molecular mechanisms that prevent central nervous system regeneration offer hope to overcome this obstacle in the future. At the same time, emergence of reprogramming of human somatic cells into pluripotent stem cells has facilitated both the generation of new source of cells with therapeutic potential and the development of innovative methods for the generation of transplantable cells. In this review, we discuss the feasibility of stem cell-based strategies applied to retinal ganglion cells and optic nerve impairment. We present the different strategies for the generation, characterization and the delivery of transplantable retinal ganglion cells derived from pluripotent stem cells. The relevance of pluripotent stem cell-derived retinal organoid and retinal ganglion cells for disease modeling or drug screening will be also introduced in the context of optic neuropathies.
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http://dx.doi.org/10.3389/fnins.2018.00651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158340PMC
September 2018

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models.

J Vis Exp 2018 09 6(139). Epub 2018 Sep 6.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, F-75012 Paris, France;

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.
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http://dx.doi.org/10.3791/57795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235103PMC
September 2018

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

J Vis Exp 2018 09 6(139). Epub 2018 Sep 6.

U861, I-Stem, Association Française contre les Myopathies (AFM), Institut National de la Santé et de la Recherche Médicale (INSERM); U861, I-Stem, Association Française contre les Myopathies (AFM), Université Evry Val-d'Essonne (UEVE);

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.
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http://dx.doi.org/10.3791/58216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235098PMC
September 2018

Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids.

Stem Cell Reports 2018 09 9;11(3):665-680. Epub 2018 Aug 9.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, 17, Rue Moreau, Paris 75012, France. Electronic address:

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.
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http://dx.doi.org/10.1016/j.stemcr.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135113PMC
September 2018

[Treating retinal dystrophies affecting retinal pigment epithelium using tissue engineering obtained from human embryonic stem cells].

Med Sci (Paris) 2018 May 13;34(5):383-386. Epub 2018 Jun 13.

Inserm U861, I-Stem, AFM, Institute for stem cell therapy and exploration of monogenic diseases, 28, rue Henri Desbruères, 91100 Corbeil-Essonnes, France - UEVE Inserm U861, I-Stem, AFM, Institute for stem cell therapy and exploration of monogenic diseases, 28, rue Henri Desbruères, 91100 Corbeil-Essonnes, France.

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http://dx.doi.org/10.1051/medsci/20183405004DOI Listing
May 2018

Noninvasive gene delivery to foveal cones for vision restoration.

JCI Insight 2018 01 25;3(2). Epub 2018 Jan 25.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.
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http://dx.doi.org/10.1172/jci.insight.96029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821199PMC
January 2018

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

Sci Transl Med 2017 Dec;9(421)

INSERM U861, I-Stem, Association Française contre les Myopathies (AFM), Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 91100 Corbeil-Essonnes, France.

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.
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http://dx.doi.org/10.1126/scitranslmed.aai7471DOI Listing
December 2017

Further Insights into the Ciliary Gene and Protein KIZ and Its Murine Ortholog PLK1S1 Mutated in Rod-Cone Dystrophy.

Genes (Basel) 2017 Oct 18;8(10). Epub 2017 Oct 18.

Sorbonne Universités, UPMC University Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, 75012 Paris, France.

We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in , encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in . KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in , the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in -mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of mutations.
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http://dx.doi.org/10.3390/genes8100277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664127PMC
October 2017

Establishment of an induced pluripotent stem (iPS) cell line from dermal fibroblasts of an asymptomatic patient with dominant PRPF31 mutation.

Stem Cell Res 2017 12 7;25:26-29. Epub 2017 Oct 7.

Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMR_S968, CNRS UMR7210, 75012 Paris, France. Electronic address:

A human iPS cell line was generated from fibroblasts of a phenotypically unaffected patient from a family with PRPF31-associated retinitis pigmentosa (RP). The transgene-free iPS cells were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system. iPS cells contained the expected c.709-734dup substitution in exon 8 of PRPF31, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had normal karyotype. This cellular model will provide a powerful tool to study the unusual pattern of inheritance of PRPF31-associated RP.
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http://dx.doi.org/10.1016/j.scr.2017.10.007DOI Listing
December 2017

Generation of an induced pluripotent stem cell (iPSC) line from a patient with autosomal dominant retinitis pigmentosa due to a mutation in the NR2E3 gene.

Stem Cell Res 2017 10 5;24:1-4. Epub 2017 Aug 5.

Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMR_S968, CNRS UMR7210, 75012 Paris, France. Electronic address:

A human iPSC line was generated from fibroblasts of a patient affected with autosomal dominant Retinitis Pigmentosa (RP) carrying the mutation p.Gly56Arg in the NR2E3 gene. The transgene-free iPSCs were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system. iPSCs contained the expected c.166G>A substitution in exon 2 of NR2E3, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had normal karyotype. This cellular model will provide a powerful tool to study the pathogenesis of NR2E3-associated RP. Resource table.
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http://dx.doi.org/10.1016/j.scr.2017.08.003DOI Listing
October 2017

Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation.

Mol Ther 2018 01 8;26(1):219-237. Epub 2017 Sep 8.

INSERM, U968, Paris 75012, France; Sorbonne Universités, UPMC Univ Paris 06 UMR_S 968, Institut de la Vision, Paris 75012, France; CNRS, UMR_7210, Paris 75012, France. Electronic address:

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.
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http://dx.doi.org/10.1016/j.ymthe.2017.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762984PMC
January 2018

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

Stem Cells 2017 05 20;35(5):1176-1188. Epub 2017 Feb 20.

Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.
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http://dx.doi.org/10.1002/stem.2586DOI Listing
May 2017

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α.

Aging Cell 2017 Feb 22;16(1):173-182. Epub 2016 Sep 22.

Institut de la Vision, 17 rue Moreau, 75012, Paris, France.

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14 blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human-induced pluripotent stem cell-derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF-α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF-α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age-related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.
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http://dx.doi.org/10.1111/acel.12540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242302PMC
February 2017

Let There Be Light: Gene and Cell Therapy for Blindness.

Hum Gene Ther 2016 Feb;27(2):134-47

1 Sorbonne Universités, UPMC Université Paris 06, INSERM, CNRS, Institut de la Vision , France .

Retinal degenerative diseases are a leading cause of irreversible blindness. Retinal cell death is the main cause of vision loss in genetic disorders such as retinitis pigmentosa, Stargardt disease, and Leber congenital amaurosis, as well as in complex age-related diseases such as age-related macular degeneration. For these blinding conditions, gene and cell therapy approaches offer therapeutic intervention at various disease stages. The present review outlines advances in therapies for retinal degenerative disease, focusing on the progress and challenges in the development and clinical translation of gene and cell therapies. A significant body of preclinical evidence and initial clinical results pave the way for further development of these cutting edge treatments for patients with retinal degenerative disorders.
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http://dx.doi.org/10.1089/hum.2015.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779297PMC
February 2016