Modélisation pathologique de l'amaurose congénitale de Leber fondée sur l'utilisation de cellules souches pluripotentes induites

L amaurose congénitale de Leber (ACL) est une maladie génétique touchant la rétine. Les premiers symptômes apparaissent dès les premiers mois de la vie et mènent en quelques années à la cécité. A ce jour, des mutations dans 18 gènes ont été associées à la maladie. Cette hétérogénéité génétique rend difficile l étude des mécanismes conduisant aux différents symptômes. Les modèles animaux utilisés en laboratoire, notamment les rongeurs, permettent d étudier certains de ces mécanismes mais présentent des limites liées à l espèce. Les cellules souches pluripotentes induites (iPSCs), qui proviennent de la reprogrammation de cellules somatiques issues de patients, constituent un nouvel outil pour étudier une maladie génétique dans un contexte humain naturel. Elles permettent d obtenir tous les phénotypes cellulaires désirés sans limite quantitative ce qui ouvre la porte à des approches d analyse à large échelle telle que l analyse transcriptomique qui vise à explorer de manière systématique la modulation des gènes dans une maladie. L objectif de mon projet de recherche a été de développer un modèle cellulaire humain naturellement porteur de l ACL. Après avoir produit les iPSCs à partir de fibroblastes de patients, mes travaux ont consisté à les différencier en populations cellulaires homogènes et facilement amplifiables, les cellules souches neurales et les cellules de l épithélium pigmentaire rétinien. Ces populations ont servi à mener des analyses transcriptomiques à large échelle qui ont permis d identifier plusieurs gènes candidats, potentiellement impliqués dans le développement de la pathologie, parmi lesquels GSTT1 qui pourrait avoir un rôle dans le stress oxydatif.Leber congenital amaurosis (LCA) is a genetic disease affecting the retina. The first symptoms appear in the first months of life and lead in few years to blindness. To date, mutations in 18 genes have been associated with the disease. This genetic heterogeneity makes it difficult to study mechanisms leading to different symptoms. Animal models, including rodents, are used to study some of these mechanisms but have limitations mostly related to the species. The induced pluripotent stem cells (iPSCs), which are reprogrammed somatic cells of patients, constitute a new tool for studying genetic diseases in a natural human context. They achieve all desired cell phenotypes without quantitative limits which opens the door to large-scale analysis approaches such as transcriptomic analysis that aims to systematically explore the modulation of genes in a disease. The aim of my research project was to develop a human cell model naturally carries the LCA. After producing the iPSCs from fibroblasts of patients, my work had consisted to differentiate them into homogeneous and easily amplifiable cell populations, neural stem cells and retinal pigment epithelial cells. These populations have served to conduct large-scale transcriptomic analyzes which have identified several candidate genes potentially involved in the development of the disease, including GSTT1 which might have a role in oxidative stress.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is characterized by retinal pigment epithelial (RPE) cell dysfunction beginning at early stages of the disease. The lack of an appropriate in vitro model is a major limitation in understanding the mechanisms leading to the occurrence of AMD. This study compared human-induced pluripotent stem cell- (hiPSC-) RPE cells derived from atrophic AMD patients () to hiPSC-RPE cells derived from healthy elderly individuals with no drusen or pigmentary alteration (). Control and AMD hiPSC-RPE cell lines were characterized by immunofluorescence, flow cytometry, and electronic microscopy. The toxicity level of iron after Fe-NTA treatment was evaluated by an MTT test and by the detection of dichloro-dihydro-fluorescein diacetate. Twelve hiPSC-RPE cell lines (6 AMD and 6 controls) were used for the experiment. Under basal conditions, all hiPSC-RPE cells expressed a phenotypic profile of senescent cells with rounded mitochondria at passage 2. However, the treatment with Fe-NTA induced higher reactive oxygen species production and cell death in hiPSC-RPE AMD cells than in hiPSC-RPE Control cells. Interestingly, functional analysis showed differences in lysosomal activity between the two populations. Indeed, Cathepsin B activity was higher in hiPSC-RPE AMD cells compared to hiPSC-RPE Control cells in basal condition and link to a pH more acidic in this cell population. Moreover, oxidative stress exposure leads to an increase of Cathepsin D immature form levels in both populations, but in a higher proportion in hiPSC-RPE AMD cells. These findings could demonstrate that hiPSC-RPE AMD cells have a typical disease phenotype compared to hiPSC-RPE Control cells

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

BACKGROUND: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. RESULTS: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. CONCLUSIONS: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF

APPROCHE DES ROLES BIOLOGIQUES DU CILIARY NEUROTROPHIC FACTOR (CNTF) DANS LE SYSTEME NERVEUX CENTRAL

LE CILIARY NEUROTROPHIC FACTOR (CNTF) EST UNE CYTOKINE NEUROPOIETIQUE APPARTENANT A LA FAMILLE DE L'INTERLEUKINE-6 (IL-6). LES CYTOKINES DE CETTE FAMILLE PARTAGENT LA MEME SOUS-UNITE RECEPTRICE : LA SOUS-UNITE GP130 ET PRESENTENT DES PROPRIETES BIOLOGIQUES COMMUNES. SI LE ROLE NEUROPROTECTEUR DU CNTF A ETE LONGUEMENT DECRIT, SES AUTRES ROLES BIOLOGIQUES DANS LE SYSTEME NERVEUX CENTRAL (SNC) SONT ENCORE MAL CONNUS. NOUS AVONS PROPOSE DANS CETTE ETUDE UNE APPROCHE DES ROLES BIOLOGIQUES DU CNTF DANS LE SNC. LE CNTF AGIT SUR LES ASTROCYTES IMMATURES QUI POSSEDENT LE RECEPTEUR TRIMERIQUE COMPOSE DU RECEPTEUR AU LEUKEMIA INHIBITORY FACTOR (LIFR) ET DE LA SOUS-UNITE CNTFR. PAR CONTRE, LES ASTROCYTES MATURES N'EXPRIMENT PAS DE FACON SIGNIFICATIVE CETTE SOUS-UNITE, ALORS QU'ILS REPONDENT A UNE ADMINISTRATION DE CNTF IN VIVO. DANS LA PREMIERE PARTIE DE L'ETUDE NOUS AVONS TENTER DE REPONDRE A CETTE CONTROVERSE EN PROPOSANT QUE LE CNTF POUVAIT AGIR SUR LES ASTROCYTES EN SE LIANT, DE FACON ILLEGITIME SUR LE RECEPTEUR DU LIF, PUISQU'IL SEMBLE QUE LES ASTROCYTES MATURES PERDENT LA SOUS-UNITE CNTFR DURANT LEUR DIFFERENCIATION. UN CERTAIN NOMBRE DE NOS RESULTATS VONT EFFECTIVEMENT DANS CE SENS, PUISQUE NOUS AVONS MONTRE QUE LES EFFETS DU CNTF SUR LES ASTROCYTES MATURES IN VITRO : (I)NECESSITAIENT UNE FORTE CONCENTRATION DU FACTEUR, (II) QU'ILS ACTIVAIENT LES VOIES DE SIGNALISATION JAK/STAT ET RAS-MAPK ; (III) QU'ILS ETAIENT PRESERVES DANS DES CELLULES CNTFR-/- ; (IV) QU'ILS ETAIENT POTENTIALISES PAR ADDITION DE CNTFR SOLUBLE DANS LE MILIEU DE CULTURE ; (V) QU'ILS ETAIENT DEPENDANTS DE L'ACTIVATION DU LIFR. SUR LA BASE DE CES RESULTATS, NOUS AVONS ETABLIS UN SCHEMA MECANISTIQUE DANS LEQUEL, LE CNTF POURRAIT INTERVENIR COMME UNE MOLECULE DE SIGNALISATION INTERGLIALE SOUS LE CONTROLE DES NEURONES. CEPENDANT, LE PROBLEME DE LA LIBERATION DU CNTF, QUI NE POSSEDE PAS DE PEPTIDE SIGNAL, HORS DE SES CELLULES PRODUCTRICES RESTAIT ENTIER. DES ETUDES RECENTES ONT MONTRE QUE LA P-GLYCOPROTEINE (P-GP), UN TRANSPORTER DE LA FAMILLE DES ABC-PROTEINES CODEES PAR LE GENE MDR (MULTIDRUG RESISTANCE), ETAIT PRESENTE ET INDUCTIBLE LORS D'UN STRESS SUR LES PIEDS ASTROCYTAIRES. DE MANIERE A ETUDIER LES ROLES POTENTIELS DE CETTE PROTEINE DANS LES ASTROCYTES, NOUS AVONS TENTE DE REGULER LA P-GP DANS UNE CULTURE ASTROCYTAIRE MENEE A LONG-TERME, DANS LAQUELLE CES CELLULES PRESENTENT UN PHENOTYPE DE CELLULE MATURE. A NOTRE ETONNEMENT, LA PLUPART DES FACTEURS CONNUS POUR INDUIRE UNE REACTIVITE ASTROCYTAIRE N'AVAIENT AUCUN EFFET SUR L'EXPRESSION DE LA P-GP. PARMI CES MOLECULES, SEULES CELLES DE LA FAMILLE DE L'IL-6 (IL-6, LIF, CT-1 ET CNTF) INDUISAIENT UNE SUREXPRESSION DE CETTE PROTEINE. CES EFFETS ETANT ABOLIS PAR L'UTILISATION DE BLOQUANTS SPECIFIQUES DE LA P-GP, CELA SUGGERE QU'ILS ETAIENT RELIES PLUS A UN EFFET POST-TRADUCTIONNEL QU'A UN EFFET TRANSCRIPTIONNEL. CETTE FAMILLE DE CYTOKINES ETANT CONNUE POUR STIMULER L'EXPRESSION DU CNTF ENDOGENE, NOUS AVONS SUGGERER QUE CETTE AUGMENTATION POUVAIT ETRE LE MECANISME COMMUN IMPLIQUE DANS LA SUREXPRESSION DE LA P-GP. NOUS AVONS VERIFIE CETTE HYPOTHESE SUR DES ASTROCYTES CNTF-/-. DANS CE CAS, LES EFFETS DES CYTOKINES, A PART CELUI DU CNTF LUI-MEME QUI PEUT AFFECTER L'EXPRESSION DE LA P-GP SUITE A SON INTERNALISATION DANS LES CELLULES, SONT ABOLIS. A LA SUITE DE CES RESULTATS, NOUS AVONS SUGGERE QUE LE MECANISME DE L'INDUCTION DE LA P-GP SUR LES ASTROCYTES ETAIT DIRECTEMENT RELIE A L'EXPRESSION DU CNTF, ET QUE LE BUT FINAL DE CETTE SUREXPRESSION, POURRAIT ETRE LA LIBERATION DU CNTF HORS DES ASTROCYTES.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Cell Therapy for Retinal Dystrophies: From Cell Suspension Formulation to Complex Retinal Tissue Bioengineering

International audienceRetinal degeneration is an irreversible phenomenon caused by various disease conditions including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). During the course of these diseases, photoreceptors (PRs) are susceptible to degeneration due to their malfunctions or to a primary dysfunction of the retinal pigment epithelium (RPE). Once lost, these cells could not be endogenously regenerated in humans, and cell therapy to replace the lost cells is one of the promising strategies to recover vision. Depending on the nature of the primary defect and the stage of the disease, RPE cells, PRs, or both might be transplanted to achieve therapeutic effects. We describe in this review the current knowledge and recent progress to develop such approaches. The different cell sources proposed for cell therapy including human pluripotent stem cells are presented with their advantages and limits. Another critical aspect described herein is the pharmaceutical formulation of the end product to be delivered into the eye of patients. Finally, we also outline the future research directions in order to develop a complex multilayered retinal tissue for end-stage patients

Challenges of cell therapies for retinal diseases

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Stem Cell-Based RPE Therapy for Retinal Diseases: Engineering 3D Tissues Amenable for Regenerative Medicine

International audienceRecent clinical trials based on human pluripotent stem cell-derived retinal pigment epithelium cells (hPSC-RPE cells) were clearly a success regarding safety outcomes. However the delivery strategy of a cell suspension, while being a smart implementation of a cell therapy, might not be sufficient to achieve the best results. More complex reconstructed tissue formulations are required, both to improve functionality and to target pathological conditions with altered Bruch's membrane like age-related macular degeneration (AMD). Herein, we describe the various options regarding the stem cell source choices and the different strategies elaborated in the recent years to develop engineered RPE sheets amenable for regenerative therapies

Photoreceptor Cell Replacement Using Pluripotent Stem Cells: Current Knowledge and Remaining Questions

International audienceRetinal degeneration is an increasing global burden without cure for the majority of patients. Once retinal cells have degenerated, vision is permanently lost. Different strategies have been developed in the recent years to prevent retinal degeneration or to restore sight (e.g., gene therapy, cell therapy and electronic implants). Herein, we present current treatment strategies with a focus on cell therapy for photoreceptor replacement using human pluripotent stem cells. We will describe the state of the art and discuss obstacles and limitations observed in preclinical animal models as well as future directions to improve graft integration and functionality

Validation of the l-dopa-induced dyskinesia in the 6-OHDA model and evaluation of the effects of selective dopamine receptor agonists and antagonists

Current treatments for Parkinson's disease (PD) rely on a dopamine replacement strategy and are reasonably effective, particularly in the early stages of the disease. However, chronic dopaminergic therapy is limited by the development of a range of side effects, including dyskinesia. This has led to a search for alternative treatments. Transplantation of foetal nigral dopamine neurons is a rational approach and many studies have shown that it can improve motor functions in parkinsonian rodents, primates and man. Recently, however, two clinical trials have reported an exacerbation of dyskinesias in some transplanted patients, raising concerns about the safety of the transplantation strategy. To study this issue, we have reproduced the l-dopa-induced dyskinesia model developed by Cenci et al. [M.A. Cenci, C.S. Lee, A. Bjorklund, l-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA, Eur. J. Neurosci. 10 (1998) 2694–2706] in the rat. We find that their abnormal involuntary movements rating scale is easy to apply and consistent to use. Moreover, the Schallert forelimb placing test has been used to assess l-dopa-induced recovery of function and we find that the rats continue to show good recovery on this test, even while they are exhibiting abnormal dyskinetic side effects. To further evaluate this model, we have studied the effects of selective dopamine receptor antagonists and agonists for D1, D2 and D3 receptors. Antagonists of all three receptors are able to block the l-dopa-induced dyskinesia without interfering with the beneficial effects of l-dopa on the placing test. This indicates that the effects of chronic l-dopa on recovery of parkinsonian symptoms and on induction of dyskinetic side effects can be dissociated, which may provide the basis for developing novel combination treatments, e.g. using grafts while blocking the unwanted adverse effects of the drugs