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Summary There is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases., Graphical Abstract, Highlights • Myotonic dystrophy type 1 hPSCs were adapted for high content screening • FDA-approved cardiac glycosides normalize in vitro and in vivo DM1 biological markers • Cardiac glycosides synergize with the ERK pathway to normalize DM1 biomarkers • This study emphasizes the value of human pluripotent stem cells for drug discovery, Physiology; Molecular Biology; Cell Biology

Wnt-ligands are among key morphogens that mediate patterning of the anterior territories of the developing brain in mammals. We qualified the role of Wnt-signals in regional specification and subregional organization of the human telencephalon using human pluripotent stem cells (hPSCs). One step neural conversion of hPSCs using SMAD inhibitors leads to progenitors with a default rostral identity. It provides an ideal biological substrate for investigating the role of Wnt signaling in both anteroposterior and dorso-ventral processes. Challenging hPSC-neural derivatives with Wnt-antagonists, alone or combined with sonic hedgehog (Shh), we found that Wnt-inhibition promote both telencephalic specification and ventral patterning of telencephalic neural precursors in a dose-dependent manner. Using optimal Wnt-antagonist and Shh-agonist signals we produced human ventral-telencephalic precursors, committed to differentiation into striatal projection neurons both in vitro and in vivo after homotypic transplantation in quinolinate-lesioned rats. This study indicates that sequentially organized Wnt-signals play a key role in the development of human ventral telencephalic territories from which the striatum arise. In addition, the optimized production of hPSC-derived striatal cells described here offers a relevant biological resource for exploring and curing Huntington disease.

Considerable hope surrounds the use of disease-specific pluripotent stem cells to generate models of human disease allowing exploration of pathological mechanisms and search for new treatments. Disease-specific human embryonic stem cells were the first to provide a useful source for studying certain disease states. The recent demonstration that human somatic cells, derived from readily accessible tissue such as skin or blood, can be converted to embryonic-like induced pluripotent stem cells (hiPSCs) has opened new perspectives for modelling and understanding a larger number of human pathologies. In this review, we examine the opportunities and challenges for the use of disease-specific pluripotent stem cells in disease modelling and drug screening. Progress in these areas will substantially accelerate effective application of disease-specific human pluripotent stem cells for drug screening.

Considerable hope surrounds the use of disease-specific pluripotent stem cells, which can differentiate into any cell type, as starting materials to generate models of human disease that will allow exploration of pathological mechanisms and the search for new treatments. Disease-specific human embryonic stem cells have provided a useful source for studying certain disease states. However, reprogramming of human somatic cells that use readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells has opened new perspectives for modeling and understanding a larger number of human pathologies. Here, we examine the challenges in creating a disease model from human pluripotent stem cells, and describe their use to model both cell-autonomous and non-cell-autonomous mechanisms, the need for adequate control experiments and the genetic limitations of human induced pluripotent stem cells. Progress in these areas will substantially accelerate effective application of disease-specific human pluripotent stem cells for drug screening.

International audience; Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.

MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS - Suppressor Target Screen – approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.

Patients with Myotonic Dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among those are cognitive dysfunctions, the origin of which has remained elusive due in part to the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis as mutant gene-carriers, in order to differentiate cells along the neural lineage. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, a RNA-binding protein whose function is defective in DM1 patients, resulted in the mTOR signaling alteration whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.

SummaryOne puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage.

The present work on group testing describes the reliable detection by ELISA of pea seed-borne mosaic virus (PSbMV) in pea seed lots. In the cultivars studied, the percentage of embryos that were ELISA-positive was correlated with the percentage of seed transmission. But unlike the seed-borne lettuce mosaic virus/lettuce and soybean mosaic virus/soybean systems, when whole pea seed was blended, the assays for PSbMV-infected seed in the experimental lot greatly overestimated the known percentage of seed transmission, due to the extraction of non-seed-transmissible virus from the seed coats. The removal of seed coats thus becomes imperative and a promising mechanical procedure is proposed for routine application. Before grouping embryos, the maximum size of a group can be determined on the basis of the variation in PSbMV titer among about 20 infected embryos oP a given cultivar. In case of pea cv. Amino, the embryo with the lowest virus titer could be detected when mixed with a maximum of 100 healthy embryos. In case of pea cv. Arpad the group size limit proposed is 60 embryos. This limit may thus vary among cultivars and antisera ; a workable limit should therefore be determined for routine use., L’objectif de ce travail est de définir les modalités de traitement de l’échantillon de graines qui permettent d’utiliser la technique ELISA pour la détermination du taux de transmission du pea seed-borne mosaic virus (PSbMV) sur différents lots de graines de Pois. Pour les cultivars étudiés, une bonne corrélation existe entre le pourcentage de transmission et le pourcentage d’embryons qui réagissent positivement au test ELISA. Mais à la différence des observations faites sur graines de Laitue lorsqu’on dose le virus de la Mosaïque de la Laitue ou sur graines de Soja lorsqu’on évalue la transmission du virus de la Mosaïque du Soja, si l’on pratique le test ELISA sur des suspensions résultant du broyage de l’ensemble de la graine avec un homogénéiseur, le pourcentage de graines infectées dépasse très largement le pourcentage de transmission du lot, car, dans ces conditions de broyage, l’antigène présent dans les téguments est lui aussi extrait. Il est donc nécessaire d’éliminer ces téguments avant le test et un procédé mécanique est proposé pour le traitement d’un nombre important de graines dans les conditions de routine. Avant de procéder au test sur les embryons ainsi obtenus, il faut déterminer le nombre maximum d’embryons sains qui, ajoutés à un embryon infecté, forment un groupe positif en ELISA. Cette taille limite a été évaluée à 60 embryons pour le cv. « Arpad », et à 100 embryons pour le cv. « Amino » sur la base d’une étude de variabilité de la concentration en virus sur une vingtaine d’embryons infectés. Avec un antiserum donné, cette limite peut être déterminée de façon simple pour un cultivar considéré. Les problèmes de l’élimination des téguments et de la détermination de la taille du groupe d’embryons étant résolus, la technique ELISA se prête avec précision et reproductibilité à l’analyse de routine des lots de graines de Pois pour le taux de transmission du PSbMV.

SummaryMyotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.

Pea seed-borne mosaic virus (PSbMV), an economically significant seed-transmitted virus of pea has been commonly found in pea germplasm collections of many countries. The virus is suspected to have spread world-wide due to the exchange of infected germplasm material. Owing to the secondary spread of the virus in the field by aphid vectors, a low level of seed infection leads to a high proportion of the disease. The symptoms produced on sensitive pea cultivars are often not very characteristic. Resistance to this virus, known to be governed by a recessive gene ’sbm’, is in the process of being incorporated into improved pea varieties. Before accomplishing this genetic programme, testing of seed lots by Enzyme Linked Immunosorbent Assay can greatly contribute in limiting the spread of the virus. In view of the importance of the disease and of the area of pea under cultivation, the present review has sought to deal at length with diverse aspects of the disease and the virus and also the sources and inheritance of resistance to PSbMV in pea germplasm collections., a récente augmentation des surfaces réservées à la production de Pois protéagineux et la fréquente contamination des collections de gènes par un virus transmissible par les graines, le Pea seed-borne mosaic virus ont conduit à compiler l’ensemble de l’information disponible à ce jour sur ce virus, d’autant que la difficulté à voir les symptômes et l’efficacité avec laquelle les pucerons transmettent le Pea seed-borne mosaic virus sont deux facteurs très favorables à sa dissémination. L’influence de ce virus sur le rendement est notable. Un gène de résistance « sbm » est en cours d’incorporation dans des variétés améliorées. Avant l’aboutissement de ce programme génétique, un contrôle des lots de semences par le test ELISA peut limiter efficacement la dissémination du Pea seed-borne mosaic virus dans les cultures de Pois.

Une expérimentation a été conduite sur 3 cultivars de pois pour apprécier l’effet du pea seed-borne mosaic virus sur le rendement. L’infection précoce a entraîné un retard de maturation d’environ 1 mois. Les graines, récoltées à maturité, étaient plus petites : une diminution du poids de 1 000 graines de 16 p. 100 a été enregistrée pour les cvs. Belinda et Finale en 1986 et de 28 p. 100 pour le cv. Amino, en 1984. Dans ce dernier cas, une augmentation du nombre de gousses - et de graines - sur les plantes infectées a partiellement compensé l’effet de la composante « calibre des graines » sur le rendement. Pour ces 3 variétés, les graines de petit calibre transmettent le PSbMV avec une fréquence notablement plus élevée. Toutefois, l’élimination de ces graines de petit calibre ne suffirait pas à éradiquer l’inoculum primaire dans des lots de semences infectées., In order to study the effect of PSbMV on yield, three commercial pea varieties were mechanically inoculated in the field. Early infection caused a delay of 4 to 5 weeks in maturity of the plants. Seeds harvested at maturity from the infected plants were smaller in diameter : a reduction in 1 000-grain weight of the order of l6 % was recorded for cvs. Belinda and Finale in 1986, and of 28 % for cv. Amino in 1984. In the latter case, the heavy reduction in grain weight was found to be partially compensated by the increase in number of pods produced per plant. In addition, in all three cultivars, small seeds were found to transmit PSbMV at a rate markedly higher than those of average and large seeds.

National audience; The work on group testing described here is complementary to experiments reported previously (MAURY et al., 1983) dealing with the potential of ELISA in testing soybean seed for soybean mosaic virus. The biological part of this paper (part I) shows that group testing gave similar results when the determination of the percentage of transmission was done using, in a comparative way, groups of 30 embryos, groups of 30 axes or groups of 30 seeds. Whatever the number of infected testas in the groups of 30 seeds, it was possible to prevent the viral antigen from testas from altering the results : indeed, after soaking seeds and grinding them briefly in a blendor, it was observed that only the embryos were ground. These results show that the procedure is practical for routine use. Part II gives mathematical elements allowing an estimate of the percentage of transmission with confidence intervals. Charts enable one to read these values directly for n = 30 seeds per group and N = 10, 30, 60 and 200 groups. An aim of this part was also to guide a good choice of n and N and to plan analyses. These mathematical data could potentially be used for group analysis with any other biological material.; Le travail présenté dans cette publication est complémentaire d’expériences rapportées précédemment (MAURY et al., 1983) sur les potentialités de la technique ELISA pour évaluer l’infection de la semence de soja par le virus de la mosaïque du soja. Dans la partie I, il est montré que l’analyse par groupes donne, dans les conditions décrites, des résultats similaires selon que la détermination du pourcentage de transmission est effectuée en utilisant, de façon comparée, des groupes de 30 embryons, des groupes de 30 axes embryonnaires ou des groupes de 30 graines. L’antigène des téguments, qui n’est pas impliqué dans le phénomène de transmission par la graine, ne doit pas être pris en compte dans le test. La phase d’obtention des embryons avant le test est rendue inutile par l’observation suivante : lors d’une brève homogénéisation de graines préalablement gonflées, les embryons, plus friables, sont broyés différentiellement ; les téguments, à peine lacérés, ne libèrent pas leur antigène dans le tampon d’extraction. Cette observation rend le test utilisable dans la routine. La partie II de cette publication donne des éléments mathématiques nécessaires à l’estimation du pourcentage de transmission dans la population de graines en fonction du nombre de groupes qui réagissent positivement en ELISA. Ce résultat peut être directement lu sur des abaques qui portent aussi les intervalles de confiance aux niveaux 0,90 ; 0,95 ; 0,99 ; 0,999. A titre d’exemple sont tracés les abaques pour n = 30 graines par groupe et N = 10, 30, 60 et 200 groupes qui se rapportent au système virus de la mosaïque du soja/semence de soja cv. « Altona ». Les abaques pour des valeurs quelconques de n et N sont aussi disponibles pour des applications de l’analyse par groupes à tout autre matériel biologique. Des conseils d’utilisation de cette méthode sont donnés ; en particulier, le lecteur est guidé pour le choix de n et de N en fonction de la précision recherchée et pour la planification de son analyse.

Pea seed borne mosaic virus (PSbMV) is a seed and aphid transmitted potyvirus of pea. It has a wide geographical distribution but it is often neglected due to lack of clear-cut symptoms of the disease in the field. Studies were therefore carried out to determine the effect of PSbMV on field peas; certain aspects of seed transmission and resistance were then analysed to critically evaluate the control strategies. The symptoms induced by the local strain PSbMV-Sv were highly dependent on temperature conditions. In the field the early inoculated pea plants exhibited a delay of 4-5 weeks for maturation. Seeds harvested at maturity from the infected plants were smaller in diameter leading to a 16% reduction in seed weight. The smaller seeds had a significantly higher seed transmission rate than the larger ones. The varietal incidence of transmission of PSbMV-Sv through seeds varied from less than one to around twelve percent in two consecutive years depending on the cultivar. In fact a bimodal seed transmission was observed, as a part from normal seed transmission, a latent strain of PSbMV, transmitted through seeds in a high frequency, was also detected in 5 week old seedlings. This strain was undetectable by ELISA in 2 week old seedlings and also in the embryos and had a very slow rate of multiplication. Besides, a new virulent pathotype, PSbMV-Pi, was identified on an Indian pea line which was found for the first time to partially overcome the monogenically recessive resistant gene sbm 1. This pathotype had a reduced multiplication on sbm 1 lines but could be easily distinguished from the local strains of the virus by its reaction on a set of 'strain differentiais'. Another strain of the same pathotype PSbMV-Pv was identified from the local isolates. The sbm 1 gene re-discovered in four Indian pea lines had a promising performance even under a heavy inoculum pressure of PSbMV in the field. The mite Tetranychus urticae Koch was found for the first time to transmit PSbMV from infected to healthy pea plants in the glasshouse., Le Pea Seed borne Mosaic Virus (PSbMV) est un potyvirus du pois transmissible par la graine et par les pucerons. Il a été détecté dans la plupart des zones de culture du pois mais son importance a été quelquefois sous-estimée en raison de l'absence de symptômes caractéristiques au champ. Pour cette raison, il était important d’obtenir quelque information relative à l'effet de ce virus sur le rendement du pois protéagineux. Un autre objectif de ce travail était de mieux connaître la transmission du PSbMV en vue d'une lutte à court terme et étudier les résistances et pathotypes pour des programmes de lutte à plus long terme. Les symptômes produits par les souches locales, PSbMV-Sv, varient avec la température. Au champ, l'infection précoce provoque un retard de maturité d'environ cinq semaines. Les graines récoltées à maturité sur les plantes infectées sont moins grosses, ce qui conduit à une perte moyenne en poids de seize pour cent. Parmi ces graines, celles de petit calibre transmettent le PSbMV-Sv avec une fréquence supérieure. Le taux variétal de transmission varie de moins de un à environ douze pour cent suivant les conditions climatiques et le cultivar. Ce résultat concerne les souches agressives du PSbMV : en effet la transmission par la graine s'est révélée bimodale, une souche faible non détectée dans les embryons apparaissant à haute fréquence après un délai de cinq semaines. Un nouveau pathotype, PSbMV-Pi, a été identifié à partir d'une lignée de pois indienne. Son taux de multiplication sur les lignées de pois possédant le gène sbm 1 est faible. Une souche locale de ce pathotype a aussi pu être sélectionnée ; elle se distingue de la souche indienne par les symptômes sur des lignées différentielles. Le gène sbm 1, retrouvé dans quatre lignées de la collection indienne, confère toutefois une bonne protection au champ sous forte pression d'inoculum. En dehors des pucerons, l'acarien Terranychus urticae Koch paraît transmettre le PSbMV.