Publications by authors named "Céline Nanteau"

10 Publications

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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

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

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

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

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

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

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

Phototoxic action spectrum on a retinal pigment epithelium model of age-related macular degeneration exposed to sunlight normalized conditions.

PLoS One 2013 23;8(8):e71398. Epub 2013 Aug 23.

Institut de la Vision, UPMC Univ Paris 06, UMR_S 968, Paris, France ; INSERM, U968, Paris, France ; CNRS, UMR_7210, Paris, France.

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071398PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751948PMC
June 2014