Publications by authors named "Gael Orieux"

12 Publications

  • Page 1 of 1

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

[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

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

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

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

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

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

Proc Natl Acad Sci U S A 2014 Jun 27;111(23):8518-23. Epub 2014 May 27.

Institut de la Vision, Institut National de la Santé et de la Recherche Médicale, U968;Sorbonne Universités, Université Pierre-et-Marie-Curie Paris 6, Unité Mixte de Recherche S968;Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7210, 75012 Paris, France;

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.
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http://dx.doi.org/10.1073/pnas.1324212111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060726PMC
June 2014

Involvement of Bcl-2-associated transcription factor 1 in the differentiation of early-born retinal cells.

J Neurosci 2014 Jan;34(4):1530-41

Institut de la Vision, INSERM UMR_S968, CNRS UMR 7210, Sorbonne Universités, UPMC Univ Paris 06, 75012 Paris, France, Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, 75 571 PARIS Cedex 12, France, CNRS-UMR 3347, INSERM U1021, Université Paris-sud11, Centre Universitaire, 91405 Orsay, France, and Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Retinal progenitor proliferation and differentiation are tightly controlled by extrinsic cues and distinctive combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we have characterized Bcl-2-associated transcription factor (Bclaf1) during rodent retinogenesis. Bclaf1 expression is restricted to early-born cell types, such as ganglion, amacrine, and horizontal cells. Analysis of developing retinas in Bclaf1-deficient mice revealed a reduction in the numbers of retinal ganglion cells, amacrine cells and horizontal cells and an increase in the numbers of cone photoreceptor precursors. Silencing of Bclaf1expression by in vitro electroporation of shRNA in embryonic retina confirmed that Bclaf1 serves to promote amacrine and horizontal cell differentiation. Misexpression of Bclaf1 in late retinal progenitors was not sufficient to directly induce the generation of amacrine and horizontal cells. Domain deletion analysis indicated that the N-terminal domain of Bclaf1 containing an arginine-serine-rich and a bZip domain is required for its effects on retinal cell differentiation. In addition, analysis revealed that Bclaf1 function occurs independently of its interaction with endogenous Bcl-2-related proteins. Altogether, our data demonstrates that Bclaf1expression in postmitotic early-born cells facilitates the differentiation of early retinal precursors into retinal ganglion cells, amacrine cells, and horizontal cells rather than into cone photoreceptors.
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http://dx.doi.org/10.1523/JNEUROSCI.3227-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705315PMC
January 2014

Behavioral changes are not directly related to striatal monoamine levels, number of nigral neurons, or dose of parkinsonian toxin MPTP in mice.

Neurobiol Dis 2003 Nov;14(2):218-28

INSERM U289, Experimental Neurology and Therapeutics, Hôpital de la Salpêtrière, 75013 Paris, France.

Behavioral analyses of mice intoxicated by the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) have generated conflicting results. We therefore analyzed the relationship between behavioral changes, loss of monoamine levels, and loss of dopaminergic cell bodies in groups of mice intoxicated with acute or subchronic MPTP protocols. Despite a higher degree of neuronal loss in the mice intoxicated using subchronic protocols, dopamine loss was severe and homogeneous in the striatum in all groups. Dopamine levels were less severely reduced in the frontal cortex in the three groups of MPTP-intoxicated mice. Norepinephrine and serotonin levels in the striatum were decreased only in the mice intoxicated with the acute protocol. The most surprising result was that the mice intoxicated with the subchronic protocols were more active than the saline-treated mice. As reported in rats with dopamine depletion in the prefrontal cortex, the hyperactivity observed in our mice could be due to the reduced dopamine levels detected in this structure.
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http://dx.doi.org/10.1016/s0969-9961(03)00108-6DOI Listing
November 2003

Levodopa but not ropinirole induces an internalization of D1 dopamine receptors in parkinsonian rats.

Mov Disord 2002 Nov;17(6):1174-9

Institut National de la Santé et de la Recherche Médicale U 289, Hôpital de la salpêtrière, Paris, France.

Levodopa therapy in Parkinson's disease is mediated by dopamine receptors and, in a recent study, we showed that a Dl full agonist can induce an internalization of D1 dopamine receptors. The aim of the present study was to determine whether levodopa or a dopamine agonist such as ropinirole can also induce the internalization of D1 dopamine receptors in the striatum of control and hemiparkinsonian rats. The distribution of D1 dopamine receptors was analyzed by immunohistochemistry using a specific antibody. In control animals and 6-hydroxydopamine (6-OHDA)-lesioned animals treated with saline, D1 dopamine receptors were localized at the level of the plasma membrane. In contrast, in both lesioned and nonlesioned animals receiving a single dose of levodopa, but not in animals receiving ropinirole, D1 dopamine receptors were internalized in the cytoplasm. This result is likely explained by the fact that ropinirole binds to non-D1 dopamine receptors, whereas levodopa, which increases dopamine levels, indirectly acts on both D1 and D2 receptors. Ropinirole is consequently less likely to desensitize D1 dopamine receptors than levodopa and, thus, to reduce the efficacy of the treatment.
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http://dx.doi.org/10.1002/mds.10256DOI Listing
November 2002

Consequences of dopaminergic denervation on the metabolic activity of the cortical neurons projecting to the subthalamic nucleus in the rat.

J Neurosci 2002 Oct;22(19):8762-70

Institut National de la Santé et de la Recherche Médicale U.289 Neurologie et Thérapeutique Expérimentale, Hôpital de la Salpêtrière, 75651 Paris Cedex 13, France.

Parkinsonian symptoms are currently thought to be related to hyperactivity of the subthalamic nucleus (STN). Because the STN is known to receive many inputs including glutamatergic cortical afferent fibers, we sought to determine whether the activity of this pathway is altered after dopaminergic denervation to estimate its contribution to the impairment of STN activity. A precise mapping of the origin of the corticosubthalamic projection was first performed using retrograde and anterograde tracing methods. Cortical neurons projecting to the STN were found to originate in layer V of the motor, anterior cingulate, and dorsal insular cortices, and the most anterior tip of the frontal lobe, leading to different functional corticosubthalamic inputs. The metabolic activity of the neurons projecting to the STN, first identified by retrograde tracing, was then evaluated by in situ hybridization of the first subunit of cytochrome oxidase (COI), a marker of metabolic activity, in unilateral 6-hydroxydopamine-lesioned rats. Measurements of COI mRNA expression showed a 38 and 41.5% decrease after dopaminergic denervation in the neurons projecting to the STN located in the motor and dorsal insular areas, respectively, whereas neuronal activity was mildly changed in neurons of the anterior cingulate cortex. The modified activity of STN neurons in parkinsonism may thus result in part from complex interactions between glutamatergic hyperactive fibers originating in the thalamus and the pedunculopontine nucleus and hypoactive fibers originating in the cerebral cortex.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757780PMC
October 2002